ES2332774T3 - 15603, A MEMBER OF THE FAMILY OF HUMAN ION CHANNELS. - Google Patents

Abstract

The invention provides isolated nucleic acids molecules, designated 15603 nucleic acid molecules, which encode novel ion channel family members. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing 15603 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a 15603 gene has been introduced or disrupted. The invention still further provides isolated 15603 proteins, fusion proteins, antigenic peptides and anti-15603 antibodies. Diagnostic and therapeutic methods utilizing compositions of the invention are also provided.

Description

15603, a member of the family of channels Ionic human.

Many intercellular events and intracellular depend on the regulation of the concentration of certain ions such as calcium, sodium, potassium and chlorine within the cell. Ionic channels that regulate flow have been discovered of anions or cations across a membrane based on the voltage, pH, phosphorylation and ligand binding. Typically, a Ionic channel consists of multiple transmembrane domains that form a channel through which ions pass from one side of the membrane to the other. These ion channels can play roles important in how a cell responds to hormones or neurotransmitters with an increase in the activity of enzymes such such as phospholipase C and a subsequent elevation in concentration of intracellular free calcium (Ca + 2). The increase in intracellular calcium concentration may occur as a result  of calcium release from intracellular reserves as well as by an entry of calcium through the plasma membrane.

It is believed that Drosophila transient receptor potential (TRP) proteins and some mammalian homologs (TRPC proteins) mediate the capacitive entry of Ca + 2 (Hofmann et al ., Nature 397: 259-263 (1999)) . Seven homologous mammalian genes (TRPC 1 to TRPC 7) have been cloned and characterized. TRPC6, together with TRPC 3, have been identified as the first members of a new functional family of cationic channels that work with a second messenger, which are activated by diacylglycerol independently of protein kinase C (Hofmann et al., Supra ). It has recently been shown that, probably, TRPC6 is the essential component of the non-selective cationic channel activated by the adrenoceptor α1 (α1-AR-NSCC), which can serve as an independent route of depletion of Ca 2+. of reservations during the increase in sympathetic activity. The androgen receptor α1 (α1-AR) is widely expressed in the vascular system. The stimulation of α1-AR leads to the activation of the C? Phospholipase coupled to the G protein, which catalyzes the formation from the phosphoinositide of 1,4,5-triphosphate (IP3) and diacylglycerol, which leads to the release of the stored Ca 2+ and the sustained input of Ca 2+. The present invention relates to a human TRP6 protein, 15603.

The present invention is based, in part, on the discovery of a new member of the family of channels ionic, referred to herein as "15603". The nucleotide sequence encoding 15603 is shown in SEQ ID No.: 1, and the amino acid sequence of a 15603 polypeptide is shown in SEQ ID NO: 2. In addition, the nucleotide sequence of the coding region is represented in SEQ ID NO: 3.

The invention provides methods to explore compounds that modulate the expression or activity of the polypeptides or nucleic acids of 15603.

In one aspect, the invention provides a method to identify a compound capable of treating a disorder angiogenic, comprising: i) contacting a compound candidate with the nucleic acid molecule of SEQ ID NO: 3 as is shown in figure 1; and ii) test the ability of the compound of inhibiting the expression of the nucleic acid molecule, identifying in this way a compound capable of treating a angiogenic disorder

In an alternative aspect, the invention provides a method to identify a compound capable of treating an angiogenic disorder, comprising: i) contacting a candidate compound with a polypeptide encoded by the molecule of nucleic acid of SEQ ID NO: 3 as shown in Figure 1 or a cell that expresses the polypeptide encoded by the molecule of nucleic acid of SEQ ID NO: 3 as shown in the figure one; and ii) test the ability of the compound to inhibit activity ion channel of the polypeptide, thus identifying a compound capable of treating an angiogenic disorder. In a embodiment, said assay is a cell based assay and said Candidate compound contacts a cell that expresses said polypeptide. In alternative embodiments, said test is A test without cells. In one embodiment, said polypeptide is provides as an ionic channel in a plasma membrane. In a embodiment, said assay of the ability of the compound to inhibit polypeptide activity comprises measuring the flow of ions to through said ion channel. In another embodiment, the ion flow through said ion channel is measured by pinch record zonal.

In one embodiment, the mentioned methods above to identify a compound capable of treating a angiogenic disorder comprise confirming the ability of a test compound inhibiting the expression of the acid molecule nucleus of SEQ ID NO: 3 as shown in Figure 1 of inhibit the polypeptide encoded by the nucleic acid molecule of SEQ ID NO: 3 as shown in figure 1 in a model animal.

Other features and advantages of the invention will be apparent from the following detailed description and to from the claims.

Brief description of the drawings

Figure 1 represents a cDNA sequence (SEQ ID NO: 1) and the predicted amino acid sequence (SEQ ID NO: 2) of human 15603 protein. The methionine initiated open reading phase of human 15603 protein is also shown (without the 5 'and 3' untranslated regions of SEQ ID NO: 1) as coding sequence, SEQ ID
Nº: 3.

       \ newpage
    

Figure 2 represents a hydropathy chart of human 15603 protein. The relatively hydrophobic remains are show above the horizontal dashed line, and the remains relatively hydrophilic are below the horizontal line of strokes Cysteine residues (cys) are indicated by short lines vertical just below the hydropathy chart. They indicated the numbers corresponding to the amino acid sequence of the Human 15603 protein - Polypeptides of the invention include fragments that include: all or part of a hydrophobic sequence, for example, a sequence above the dashed line, for example, the sequence from about amino acid 162 to 172, from about amino acid 215 to 225, from approximately amino acid 325 to 335, from about amino acid 401 to 431, from about amino acid 441 to 461, from about amino acid 486 to 506, from approximately amino acid 521 to 551, from about amino acid 591 to 616, from about amino acid 631 to 656, from about amino acid 665 to 675, from about amino acid 701 to 731, and from about amino acid 770 to 780 of SEQ ID NO: 2; all or part of a hydrophilic sequence, for example, a sequence below the dashed line, for example, the sequence from approximately the amino acid 20 to 32, from about amino acid 55 to 85, from about amino acid 181 to 221, from approximately amino acid 241 to 281, from about amino acid 306 to 321, from about amino acid 331 to 340, from about amino acid 461 to 486, from approximately amino acid 780 to 805, from about amino acid 810 to 841, from about amino acid 841 to 851, from about amino acid 865 to 890, from approximately amino acid 895 to 905, and from about amino acid 915 to 931 of SEQ ID NO: 2; and a sequence that It includes a Cys, or a glycosylation site.

Figure 3 represents an alignment of the ion transport protein and repeat domains of ank of the human 15603 protein with a consensus amino acid sequence from a hidden Markov model (HMM) from PFAM. The Upper sequence is the consensus amino acid sequence (SEQ ID No.: 4,5,6) for an ank repeat domain, while the lower amino acid sequence corresponds to amino acids 97 to 131, 132 to 163 and 218 to 250 of SEQ ID NO: 2. Also shown The consensus amino acid sequence (SEQ ID NO: 7) for a domain of ionic transport protein aligned with amino acids 493 a 727 of SEQ ID NO: 2.

Figure 4 represents a BLAST alignment of the ion channel domain of human 15603 protein with a consensus amino acid sequence of a base derived domain ProDomain ("Channel Transient Repeat Calcium Receiver) Potential Ion Ank Transport Transmembrane "; No. PD004194; ProDomain Release 2001.1; http://www.toulouse.inra.fr/prodom.html). The lower sequence is amino acid residues 52 to 278 of the PD004194 consensus sequence of 278 amino acids (SEQ ID NO: 8), while the upper amino acid sequence corresponds to Ionic channel domain of human 15603 protein, residues amino acids 123 to 347 of SEQ ID NO: 2.

Detailed description of the invention

The sequence of human 15603 protein (Figure one; SEQ ID NO: 1), which is approximately 2796 long nucleotides including untranslated regions, contains a expected methionine initiated coding sequence of approximately 2796 nucleotides, including termination codon (nucleotides indicated as "encoders" of SEQ ID NO: 1 in Fig. 1; SEQ ID NO: 3). The coding sequence encodes a 931 amino acid protein (SEQ ID NO: 2). 15603 protein human of SEQ ID NO: 2 and Figure 2 includes a sequence of amino-terminal hydrophobic amino acids, consistent with a signal sequence of approximately 26 amino acids (from the amino acid 1 to about amino acid 26 of SEQ ID NO: 2, PSORT, Nakai and Kanehisa (1992) Genomics 14: 897-911), which after excision provides a form of mature protein.)). This form of mature protein It has a length of approximately 905 amino acid residues (from approximately amino acid 27 to amino acid 931 of SEQ ID NO: 2).

Human 15603 protein contains the following regions or other structural features (for general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, see Sonnhammer et al . (1997) Protein 28: 405- 420 and http://www.psc.edu/general/ software / packages / pfam / pfam.html:

an ank domain (PFAM Access Number PF0023) located approximately in amino acids 97 to 131 of SEQ ID NO: 2;

an ank domain (PFAM Access Number PF0023) located approximately at amino acids 132 to 163 of SEQ ID NO: 2;

an ank domain (PFAM Access Number PF0023) located approximately at amino acids 218 to 250 of SEQ ID NO: 2;

an ionic transport protein domain (PFAM Access Number PF00520) located approximately at amino acid residues 493 to 727 of SEQ ID NO: 2;

an ionic calcium entry domain of transient potential repeating variant of the receiving channel (ProDom No. PD140156) located approximately in the remains amino acids 1 to 39 of SEQ ID NO: 2;

an ionic calcium entry domain of transient potential repeating variant of the receiving channel (ProDom No. PD323618) located approximately in the remains amino acids 40 to 71 of SEQ ID NO: 2;

a related repeat variant domain with Trp of capacitive calcium input of transient potential of receiver channel (ProDom No. PD186301) located approximately at amino acid residues 93 to 133 of SEQ ID NO: 2;

an ank domain of Trp Vision transient potential for ionic receptor repeat channel (ProDom No.
PD140149) located approximately at amino acid residues 95 to 154 of SEQ ID NO: 2;

a factor of the type of the recipient family of ankyrin with ORF of the repeated ankyrin channel gene (ProDom No. PD007334) located approximately in amino acid residues 102 to 166 is SEQ ID NO: 2;

an alternative EGF glycoprotein domain related to ankirine UNC-44, kinase domain of ankirine repeat (ProDom No. PD000041) located approximately at amino acid residues 105 to 188 of SEQ ID Nº: 2;

a transmembrane domain of transport ank ionic calcium repeating channel transient potential receiver (ProDom No. PD004194) located approximately in the amino acid residues 123 to 347 of SEQ ID NO: 2;

a NOMPC TRP2 domain Y71A12B.4 Y71A12B.E 2-Beta 2-Alpha of transient potential of the receptor channel (ProDom No. PD296552) located approximately in amino acid residues 299 to 509 of SEQ ID
Nº: 2;

an MTR1 domain of the Fis Transmembrane Receiver (ProDom No. PD039592) located approximately in the remains amino acids 308 to 916 of SEQ ID NO: 2;

a transmembrane calcium ion transport domain of transient potential of receptor channel repeat (ProDom No. PD328255) located approximately at amino acid residues 362 to 509 of SEQ ID
Nº: 2;

a transmembrane ion transport domain of vanilloid repetition of transient channel potential calcium receptor (ProDom No. PD003230) located approximately in amino acid residues 409 to 748 of SEQ ID NO: 2;

a transient ionic variant Ank domain of repetition of calcium receptor channel potential (ProDom No. PD238062) located approximately at 510 amino acid residues to 597 of SEQ ID NO: 2;

an ionic inbound transport domain capacitive transient potential of receiver channel repetition  of calcium (ProDom No. PD342728) located approximately in the amino acid residues 744 to 895 of SEQ ID NO: 2;

a transmembrane domain of transport ank calcium ionic transient channel repeat potential receiver (ProDom No. PD004174) located approximately in the amino acid residues 749 to 900 of SEQ ID NO: 2;

a transport ank repeat domain transient ionic receiver input channel receiver calcium (ProDom No. PD266294) located approximately in the amino acid residues 786 to 854 of SEQ ID NO: 2;

a domain related to Gelsolin (ProDom No. PD202783) located approximately in the amino acid residues 792 to 931 of SEQ ID NO: 2;

a sevenfold pattern muscle domain of supercoiled myosin repeat heavy chain filament (ProDom No. PD000002) located approximately in the remains amino acids 796 to 928 of SEQ ID NO: 2;

a calcium input ion variant of repetition of transient potential of receiver channel (ProDom No. PD137340) located approximately in amino acid residues 901 to 931 of SEQ ID NO: 2;

a transmembrane domain (provided by MEMSAT, Jones et al . (1994) Biochemistry 33: 3038-3049) approximately at amino acid residues 407 to 426 of SEQ ID NO: 2;

a transmembrane domain (provided by MEMSAT, Jones et al . (1994) Biochemistry 33: 3038-3049) approximately in amino acid residues 443 to 459 of SEQ ID NO: 2;

a transmembrane domain (provided by MEMSAT, Jones et al . (1994) Biochemistry 33: 3038-3049) approximately in amino acid residues 490 to 507 of SEQ ID NO: 2;

a transmembrane domain (provided by MEMSAT, Jones et al . (1994) Biochemistry 33: 3038-3049) approximately in amino acid residues 598 to 614 of SEQ ID NO: 2;

a transmembrane domain (provided by MEMSAT, Jones et al . (1994) Biochemistry 33: 3038-3049) approximately in amino acid residues 637 to 653 of SEQ ID NO: 2;

a transmembrane domain (provided by MEMSAT, Jones et al . (1994) Biochemistry 33: 3038-3049) approximately at amino acid residues 703 to 727 of SEQ ID NO: 2;

a supercoiled structure located approximately in amino acid residues 296 to 907 (ALELSNELAV ... LEEKSQNTED) of SEQ ID NO: 2;

a leucine zipper pattern (Prosite PS00029) located approximately in amino acid residues 766 to 787 (LVPSPKSLFYLLLKLKKWISEL) of SEQ ID NO: 2;

nine protein phosphorylation sites kinase C (Prosite PS00005) located approximately in the amino acid residues 14 to 16 (SPR), 89 to 91 (SDR), 563 to 565 (TLK), 630 to 632 (TVK), 674 to 676 (SFK), 769 to 771 (SPK), 836 to 838 (SIR), 893 to 895 (SLR) and 929 to 931 (TNR) of SEQ ID NO: 2;

sixteen casein phosphorylation sites kinase II (Prosite PS00006) located approximately in the amino acid residues 96 to 99 (SIEE), 197 to 200 (SQSE), 216 to 219 (SSHD), 289 to 292 (SSED), 296 to 299 (TALE), 475 to 478 (TSTD), 492 to 495 (SWME), 556 to 559 (SIID), 563 to 566 (TLKD), 571 to 574 (TLGD), 630 to 633 (TVKD), 669 to 672 (TTVE), 730 to 733 (SFQE), 752 to 755 (SYFE), 815 to 818 (SHED) and 839 to 842 (SSED) of SEQ ID NO: 2;

two protein kinase phosphorylation sites cAMP / GMPc dependent (Prosite PS00004) located approximately in amino acid residues 67 to 70 (RRQT) and 319 to 322 (KKLS) of SEQ ID NO: 2;

three tyrosine phosphorylation sites (Prosite PS00007) located approximately in the remains amino acids 24 to 31 (RRNESQDY), 101 to 108 (RFLDAAEY) and 698 to 705 (KFIENIGY) of SEQ ID NO: 2;

two amidation sites (Prosite PS00009) located approximately in amino acid residues 75 to 78 (KGRR) and 188-191 (EGKR) of SEQ ID NO: 2;

nine N-glycosylation sites (Prosite PS00001) located approximately in the remains amino acids 26 to 29 (NESQ), 157 to 160 (NLSR), 362 to 365 (NLSR), 394 to 397 (NLSG), 473 to 476 (NETS), 561 to 564 (NDTL), 617 to 620 (NESF), 712 to 715 (NVTM) and 728 to 731 (NSSF) of SEQ ID NO: 2; Y

five sites of N-myristoylation (Prosite PS00008) located approximately in the remains amino acids 17 to 22 (GAAGAA), 213 to 218 (GTRSSH), 504 to 509 (GMIWAE), 661 to 666 (GAKQNE) and 811 to 816 (GILGSH) of SEQ ID NO: 2.

       \ vskip1.000000 \ baselineskip
    

Protein 15603 contains a number significant structural features in common with members  of the ion channel family and the repetition family of ank. The term "family", when referring to protein and nucleic acid molecules of the invention, are refers to two or more proteins or nucleic acid molecules that they have a common domain or structural motive and they have a sufficient amino acid or nucleotide sequence homology as defined herein. These family members they can be natural or unnatural and can come from it species or of different species. For example, a family can contain a first protein of human origin as well as others different proteins of human origin or, alternatively, may contain homologues of non-human origin, for example, proteins of rat or mouse Family members may also have common functional characteristics.

As used herein, the expression "ion channel" includes a protein or polypeptide that can regulate the flow of ions such as calcium cations through a channel. The channel can regulate the flow of a cation or anion particular or may be less discriminative and allow the flow to your through multiple types of cations or anions. Ion flow can be regulated by the presence or absence of a bound ligand or can be regulated by the phosphorylation state of the channel or other protein associated with the channel. The ionic channel protein can experience a conformational change based on the union of a ligand, phosphorylation of a particular moiety or the binding of another protein or biomolecule

Members of a family of proteins from Ionic channels are characterized by transmembrane domains, domains  cytoplasmic, extracellular domains, and can be homodimers, homotymers, homomultimers, heterodimers etc. The pore of the ion channels typically formed by multiple proteins transmembrane encoded by the same gene or by different genes. Typically, a hydrophilic transmembrane channel is formed that allows the passage of ions from one side of the plasma membrane to the other. Groups of amino acids loaded in the mouth of the canal may increase selectivity for a particular type of ion, for example, groups of negatively charged amino acid residues in the mouth of the canal They can exclude negative ions from ion channels.

All proteins of the family of channels Ionic form pores filled with water through the membranes. Ionic channel proteins are located in the plasma membrane of animal and plant cells and it additionally characterized by small pores of high selectivity that participate in ionic transport. Typically, more than 10 6 ions can pass through said channel every second. Many ion channels allow diffusion of specific ions such as Na +, K +, Ca 2+, or Cl -, for their electrochemical gradients through the lipid bilayer. The Ionic channel proteins show ionic selectivity, which allow some ions to pass but not others. Another feature of the ionic channel proteins is that they don't open from continuous way, unlike simple pores for the passage of Water. Instead they have "openings" that open briefly and Then they close again. This opening and closing normally responds to a specific membrane disturbance, such as a change in voltage across the membrane (opening channels by voltage), mechanical stimulation (mechanically open channels) or the binding of a signaling molecule (opening channels by ligand). In the case of ligand opening channels, the signaling ligand can be an extracellular mediator, such as a neurotransmitter (opening channels by neurotransmitter), a intracellular mediator, such as an ion (opening channels by ions), a nucleotide (nucleotide opening channels) or a GTP binding regulatory protein (opening channels by protein G).

Ionic channels are responsible for the electrical excitability of nerve and muscle cells and mediate most forms of electrical signaling in the nervous system. A single nerve cell typically contains more than five types of ion channels. However, these ion channels are not restricted to electrically excitable cells. Ionic channels are present in all animal cells and are found in plant cells and microorganisms. For example, ionic channel proteins propagate the closing response of mimosa leaves and allow the unicellular organism known as paramecium to reverse the direction after a collision. Protein 15603, also known as TRPC6, is a non-selective cation channel that is activated by diacylglycerol in a membrane-delimited manner, regardless of protein.
kinase C.

A 15603 polypeptide may include a "domain. ion channel "or homologous regions with a" channel domain ionic ". A 15603 polypeptide may further include a" domain of ank "or regions homologous to an" domain of ank ".

As used herein, the expression "ionic transport protein domain" includes a amino acid sequence of approximately 200 to 250 residues amino acids in length and having a bit score value (score in information units (bits)) for alignment of the sequence with the ionic transport protein domain (HMM) of at least 75. Preferably, a transport domain Ionic regulates the flow of ions through a membrane. Preferably, an ionic transport protein domain includes at least about 200 to 300 amino acids, more preferably from about 200 to 250 amino acid residues, or about 210 to 240 amino acids and has a bit value score for alignment of the sequence with the domain of the Ionic transport protein (HMM) of at least 50, 75, 80 or greater. The ionic transport domain can include domains transmembrane The ionic transport protein (HMM) domain is You have been assigned the PFAM Access Number PF00520 (http; // genome.wustl.edu/Pfam/.html). Figure 3 shows an alignment of the ionic transport protein domain (amino acids 493 to 727 of SEQ ID NO: 2) of polypeptide 15603 human with the consensus amino acid sequence of the protein of ionic transport of Pfam (SEQ ID NO: 7) obtained with a model hidden from Markov.

As used herein, the expression "ank repeat domain" includes a sequence of amino acids of approximately 30 to 35 amino acid residues of length and that has a bit score value for alignment of the sequence with the ank repeat domain (HMM) of at least 15. Preferably, an ank repeat domain includes the less than about 20 to 40 amino acids, more preferably of about 25 to 35 amino acid residues, or about 30 to 35 amino acids and has a bit score value for the sequence alignment with the repeat domain of ank (HMM) of at least 10, 15, 20 or greater. To the repeat domain of ank (HMM) has been assigned the PFAM Access Number PF0023 (http; // genome.wustl.edu/Pfam/.html). In addition, to the domain of ank repeat (HMM) has been assigned the SMART identifier ANK_2a (http://smart.embl-heidelberg.de/). In the Figure 3 depicts an alignment of the repeat domain of ank (amino acids 97 to 131, 132 to 163 and 218 to 250 of SEQ ID NO: 2) of human 15603 protein with the amino acid sequence Pfam ank repeat domain consensus (SEQ ID NO: 4,5,6) obtained with a hidden Markov model. Figure 3 shows represents an alignment of the repeat domain of ank (amino acids 97 to 126, 132 to 160 and 218 to 247 of SEQ ID NO: 2) of the human 15603 protein with the consensus amino acid sequence of the SMART domain ANK_2a (SEQ ID NO: 4,5,6) obtained with a model hidden from Markov.

Preferably, a 15603 polypeptide or protein has an "ionic transport protein domain" or a region that includes at least about 200 to 300 plus preferably about 200 to 250 or 210 to 240 residues amino acids and has a homology of at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% with a "protein domain of ionic transport ", for example, the protein domain of ionic transport of human 15603 protein (for example, the residues 493 to 727 of SEQ ID NO: 2).

Preferably, a 15603 polypeptide or protein has a "repeat domain of ank" or a region that includes at least about 25 to 40, more preferably of approximately 25 to 35 or 30 to 35 amino acid residues and has a homology of at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% with an "ank repeat domain", for example, an ank repeat domain of human 15603 protein (for example, residues 97 to 131, 132 to 163, or 218 to 250 of SEQ ID Nº: 2).

To identify the presence of an "ionic transport protein" domain or an "ank repeat" domain in a 15603 protein sequence, and to determine that a polypeptide or protein of interest has a particular profile, the sequence of Protein amino acids can be screened against the HMM Pfam database (for example, the Pfam database, edition 2.1) using the default parameters (http://www.sanger.ac.uk/Software / Pfam / HMM_search). For example, the hmmsf program, which is available as part of the HMMER search program package, is a family of specific default programs for MILPAT0063 and a score of 15 is the default threshold score for determining a hit (hit). Alternatively, the threshold score for determining a hit may be reduced, (for example, to 8 bits). A description of the Pfam database can be found in Sonhammer et al . (1997) Proteins 28: 405-420 and a detailed description of HMM can be found, for example, in Gribskov et al . (1990) Meth. Enzymol 183: 146-159; Gribskov et al . (1987) Proc. Natl Acad. Sci . USA 84: 4355-4358; Krogh et al . (1994) J. Mol. Biol. 235: 1501-1531; and Stultz et al . (1993) Protein Sci . 2: 305-314. A search was conducted against the HMM database that resulted in the identification of an "ionic transport protein domain" in the amino acid sequence of human protein 15603 approximately in residues 493 to 727 of SEQ ID NO: 2 (see Figure 1). A search was conducted against the HMM database that resulted in the identification of three "ank repeat" domains in the amino acid sequence of human protein 15603 approximately in residues 97 to 131, 132 to 163 and 218 to 250 of SEQ ID NO: 2 (see Figure 1).

As an additional method to identify the presence of an "ank repeat" domain in a 15603 protein sequence, and to determine that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against to a SMART database (Simple Modular Architecture Research Tool, http://smart.embl-heidelberg.de/) from HMM as described in Schultz et al . (1998), Proc. Natl Acad. Sci. USA. 95: 5857 and Schultz et al . (2000) Nucl. Acids Res 28: 231. The database contains domains identified by profiling with the hidden Markov models of the HMMer2 search program (Durbin et al . (1998) Biological sequence analysis: probabilistic models of proteins and nucleic acids . Cambridge University Press .; http://hmmer.wustl.edu/). The database is also annotated and controlled extensively by experts to improve accuracy. A search was performed against the HMM database that resulted in the identification of three "ank 2a" domains in the amino acid sequence of human 215603 protein approximately in residues 97 to 126, 132 to 160 and 218 to 247 of SEQ ID NO: 2 (see Figure 1).

For further identification of domains in a 15603 protein sequence, and to make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against a domain database, by For example, the ProDom database (Corpet et al . (1999), Nucl. Acids Res. 27: 263-267). The ProDom protein domain database consists of an automatic collection of homologous domains. Current versions of ProDom are being built using recursive PSI-BLAST searches (Altschul et al . (1997) Nucleic Acids Res. 25: 3389-3402; Gouzy et al . (1999) Computers and Chemistry 23: 333-340) of the bases of SWISS-PROT 38 and TREMBL protein data. The database automatically generates a consensus sequence for each domain. A BLAST search was performed against the HMM database which resulted in the identification of a "transmembrane repeat ion ion calcium transport transient potential transient receptor domain" in the amino acid sequence of approximately 15603 human protein in residues 123 to 347 of SEQ ID NO: 2 (see Figure 1).

A molecule of 15603 may also include a transmembrane domain or a repeat domain of ank.

A 15603 polypeptide may include at least one, two, three, four, five or preferably six "transmembrane domains" or homologous regions with "transmembrane domains." As used herein, the term "transmembrane domain" includes an amino acid sequence of approximately 10 to 40 amino acid residues in length and encompassing the plasma membrane. The transmembrane domains are rich in hydrophobic moieties, for example, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, for example, leucines, isoleucines, tyrosines or tryptophan. Transmembrane domains typically have alpha helix structures and are described, for example, in Zagotta et al ., (1996) Annual Rev. Neurosci . 19: 235-263. The transmembrane domains of human 15603 protein are located approximately at residues 407 to 426, 443 to 459,490 to 507, 598 to 614, 637 to 653 and 703 to 727 of SEQ ID NO: 2.

In a preferred embodiment, a polypeptide or 15603 protein has at least one, two, three, four, five or preferably six "transmembrane domains", or a region that includes at least about 12 to 35, more preferably of approximately 14 to 30 or 15 to 25 amino acid residues and has a homology of at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% with a "transmembrane domain", for example, transmembrane domains of human 15603 protein (for example, the residues 407 to 426, 443 to 459, 490 to 507, 598 to 614, 637 to 653, or 703 to 727 of SEQ ID NO: 2). The protein transmembrane domain 15603 human is visualized in the hydropathy chart (Figure 2) as regions of approximately 15 to 25 amino acids in which the Hydropathy chart is mainly above the line horizontal.

To identify the presence of a "transmembrane" domain in a 15603 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be analyzed by a transmembrane prediction method that predicts the secondary structure and topology of integral membrane proteins based on the recognition of topological models (MEMSAT, Jones et al ., (1994) Biochemistry 33: 3038-3049).

A 15603 polypeptide may include at least one, two, three, four, five, six or preferably seven "non-transmembrane regions." As used herein, the term "non-transmembrane region" includes an amino acid sequence not identified as a transmembrane domain. The non-transmembrane regions in protein 15603 are located approximately in amino acid residues 1 to 406, 427 to 442, 460 to 489, 508 to 597, 615 to 636, 654 to 702 and 727 to 931 of SEQ ID NO: 2. The non-transmembrane domains can be cytoplasmic or
extracellular

The non-transmembrane regions of the protein 15603 include at least one, two, three or preferably four cytoplasmic regions. When it is located at the N end, the cytoplasmic region is referred to herein as "domain N-terminal cytoplasmic. "As used in the present memory, a "cytoplasmic domain N-terminal "includes an amino acid sequence having about 1 to 500, preferably of about 1 to 450, more preferably about 1 to 425, or even more preferably about 1 to 410 amino acid residues in length, and is located inside of a cell or within the cytoplasm of a cell. The rest C-terminal amino acid of a "domain N-terminal cytoplasmic "is adjacent to a N-terminal amino acid residue of a domain transmembrane in a 15603 protein. For example, a domain N-terminal cytoplasmic is located approximately in amino acid residues 1 to 406 of SEQ ID NO: 2.

In a preferred embodiment, a polypeptide or 15603 protein has a cytoplasmic region or domain N-terminal that includes approximately 1 to 450, preferably from about 1 to 425, and more preferably of about 1 to 410 amino acid residues and has a homology of at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% with a "cytoplasmic domain N-terminal ", for example, the cytoplasmic domain N-terminal of human 15603 protein (per example, residues 1 to 406 of SEQ ID NO: 2).

In another embodiment, a cytoplasmic region of 15603 includes at least one, and preferably two loops cytoplasmic As used herein, the term "loop" includes an amino acid sequence that is not included within a phospholipid membrane, which has a length of at less about 4, preferably about 5 to 95, and more preferably about 6 to 35 residues amino acids, and has an amino acid sequence that connects two transmembrane domains within a protein or polypeptide. By consequently, the N-terminal amino acid of a loop is adjacent to a C-terminal amino acid of a transmembrane domain in a 15603 molecule, and the amino acid C-terminal of a loop is adjacent to a N-terminal amino acid of a transmembrane domain in a 15603 molecule. As used herein, a "cytoplasmic loop" includes a loop located in the inside a cell or inside the cytoplasm of a cell. By example, a "cytoplasmic loop" can be found approximately in amino acid residues 460 to 489 and 615 to 636 of SEQ ID NO: 2.

In a preferred embodiment, a polypeptide or 15603 protein has a cytoplasmic loop or a region that includes at least about 4, preferably about 5 to 40, and more preferably about 6 to 35 residues amino acids and has a homology of at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% with a "cytoplasmic loop", for example, a cytoplasmic loop of human 15603 protein (for example, residues 460 to 489 and 615 to 636 of SEQ ID NO: 2).

In another embodiment, a non-transmembrane region of 15603 includes at least one, two or preferably three loops not cytoplasmic As used herein, a "loop does not cytoplasmic "includes a loop located outside a cell or inside an intracellular organelle. Non-cytoplasmic loops include extracellular domains (i.e. outside the cell) and intracellular domains (that is, within the cell). When refers to membrane bound proteins found in intracellular organelles (e.g., mitochondria, reticulum endoplasmic, peroxisomes, microsomes, vesicles, endosomes and lysosomes), non-cytoplasmic loops include the domains of the protein that reside in the lumen of the organelle or the matrix or the intermembrane space. For example, a "loop" can be found non-cytoplasmic "approximately in amino acid residues 427 to 442, 508 to 597 and 654 to 702 of SEQ ID NO: 2.

In a preferred embodiment, a polypeptide or 15603 protein has at least one non-cytoplasmic loop or region which includes at least about 4, preferably of about 5 to 100, more preferably about 6 to 90 amino acid residues and has a homology of at least approximately 60%, 70%, 80%, 90%, 95%, 99% or 100% with a "loop non-cytoplasmic "for example, at least one non-cytoplasmic loop of human protein 15603 (for example, residues 427 to 442, 508 to 597 and 654 to 702 of SEQ ID NO: 2).

In another embodiment, a cytoplasmic region of a 15603 protein may include the C-terminus and may be a "C-terminal cytoplasmic domain" also known herein as "C-terminal cytoplasmic tail." As used herein, a "C-terminal cytoplasmic domain" includes an amino acid sequence that is at least about 200 in length, preferably about 150 to 250, more preferably about 175 to 225 amino acid residues, and is located inside a cell or inside the cytoplasm of a cell. The N-terminal amino acid residue of a "C-terminal cytoplasmic domain" is adjacent to a C-terminal amino acid residue of a transmembrane domain in a 15603 protein. For example, a C-terminal cytoplasmic domain is located approximately at amino acid residues 728 to 931 of the
SEQ ID NO: 2.

In a preferred embodiment, a polypeptide or 15603 protein has a cytoplasmic domain C-terminal or a region that includes at least about 200, preferably about 150 to 250 and more preferably from about 175 to 225 residues amino acids and has a homology of at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% with a "cytoplasmic domain C-terminal "for example, the cytoplasmic domain C-terminal of human 15603 protein (by example, residues 728 to 931 of SEQ ID NO: 2).

A human 15603 protein may also include a leucine zipper or a supercoiled structure. Preferably, a leucine zipper domain has a homology of at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% with the leucine zipper domain of 15603 protein human (for example, residues 766 to 787 of SEQ ID NO: 2). Preferably, a supercoiled domain has a homology of at least about 60%, 70%, 80%, 90%, 95%, 99% or 100% with the supercoiled domain of human 15603 protein (for example, residues 296 to 907 of SEQ ID NO: 2). A human 15603 protein may also include N-glycosylation sites, cAMP-dependent protein kinase phosphorylation sites and CGMP, protein kinase C phosphorylation sites, sites of phosphorylation of casein kinase II, phosphorylation sites of tyrosine kinase, N-myristoylation sites and Amidation sites

A family member of 15603 may include at least one ionic channel protein domain; and at least one, two or preferably three repeat domains of ank or domains transmembrane or non-transmembrane. A family member of 15603 can include at least one leucine zipper structure or at less a supercoiled structure. In addition, a member of the family of 15603 can include at least one, two, three, four, five, six, seven, eight or preferably nine sites of phosphorylation of protein kinase C (Prosite PS00005); at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen and preferably sixteen sites of phosphorylation of casein kinase II (Prosite PS00006); at least one, two, three, four, five, six, seven, eight and preferably nine N-glycosylation sites (Prosite PS00001); at least one and preferably two phosphorylation sites of cAMP / cGMP-dependent protein kinase (Prosite PS00004); to the minus one and preferably two amidation sites (Prosite PS00009); and at least one, two, three, four and preferably five N-myristoylation sites (Prosite PS00008).

As the 15603 polypeptides of the invention they can modulate activities mediated by 15603, they can be useful for the development of new diagnostic and therapeutic agents for disorders associated with ionic or other channels disorders associated with 15603 protein, as described more ahead.

As used herein, a "activity associated with ion channels" includes an activity which implies the regulation of the flow of ions through a membrane. The ion flow can be controlled for a second messenger such as diacylglycerol. Certain family members they can intervene in cardiovascular diseases such as a anomalous angiogenesis.

As used herein, an "activity of 15603", "biological activity of 15603" or "functional activity of 15603" refers to an activity exerted by a protein, polypeptide or nucleic acid molecule of 15603, for example, in a cell that responds to 15603 or in a 15603 substrate, for example a protein substrate, as determined in vivo or in vitro. In one embodiment, an activity of 15603 is a direct activity, such as an association with a target molecule of 15603. A "target molecule" or "binding partner" is a molecule with which a protein binds or interacts in nature. 15603. In an illustrative embodiment, protein 15603 is an ionic channel, for example, a non-selective cation channel that is activated by diacylglycerol, and therefore interacts in nature with a molecule such as a second messenger (for example, diacylglycerol) to regulate the flow of cations through a membrane.

An activity of 15603 can also be a indirect activity, for example, a signaling activity cell mediated by the interaction of 15603 protein with a 15603 receiver. Based on the sequence structures described above and in the similarities with molecules of known function, the 15603 molecules of the present invention they may have similar biological activities to the members of the ion channel family. For example, the 15603 proteins of the The present invention may have one or more of the following activities: (1) the ability to join a second messenger; (2) the ability to bind diacylglycerol; (3) the ability to regulate the flow of cations through a membrane; (4) the ability to regulate angiogenesis; and (5) the ability to regulate intracellular calcium levels.

The 15603 molecules of the invention can modulate the activities of cells in tissues in which express. For example, the mRNA of 15603 is expressed in levels of Moderate to elevated in hemangiomas, megakaryocytes, Wilm tumor, vascular smooth muscle cells, endothelial cells Proliferative, uterus and normal cerebral cortex. Therefore, 15603 molecules of the invention can act as agents therapeutic or diagnostic for cardiovascular disorders, angiogenic or neoproliferative.

In this way, 15603 molecules can act as new diagnostic targets and therapeutic agents to control one or more cardiovascular disorders, angiogenic, Neoproliferative or other ionic channel disorders. How I know used herein, "ion channel disorders" they are diseases or disorders whose pathogenesis is caused by, is related or associated with an aberrant function or expression or deficient protein of ion channels.

15603 molecules can be used to treat cardiovascular, immune or proliferative disorders in part because family members are found in hemangiomas, blood vessels and megakaryocytes.

As used herein, a "endothelial cell disorder" includes a disorder characterized by an activity of endothelial cells aberrant, unregulated or unwanted, for example, angiogenesis; wave aberrant expression of adhesion molecules on the surface cell or genes associated with angiogenesis, for example, TIE-2, FLT and FLK. Cell disorders Endothelial include tumorigenesis, tumor metastasis, psoriasis, diabetic retinopathy, endometriosis, Grave's disease, ischemic disease (for example, atherosclerosis) and diseases chronic inflammatory (for example, rheumatoid arthritis).

The 15603 protein, its fragments and derivatives and Other variants of the sequence in SEQ ID NO: 2 are called collectively "15603 proteins or polypeptides". Molecules of nucleic acid encoding these polypeptides and proteins are collectively called "15603 nucleic acids".

As used herein, the expression "nucleic acid molecule" includes DNA molecules (by example, a cDNA or genomic DNA) and RNA molecules (for example, a MRNA) and analogs of the DNA or RNA generated, for example, by use of nucleotide analogues. The nucleic acid molecule can be single-stranded or double-stranded, but preferably it is DNA double-stranded

The expression "nucleic acid molecule isolated or purified "includes nucleic acid molecules that are separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. For example, with with respect to genomic DNA, the term "isolated" includes nucleic acid molecules that are separated from the chromosome with which is naturally associated genomic DNA. Preferably, an "isolated" nucleic acid lacks flanking sequences naturally nucleic acid (i.e. sequences located at the 5 'and / or 3' ends of the nucleic acid) in the DNA genomic of the organism from which the nucleic acid comes. By example, in various embodiments, the nucleic acid molecule isolated may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5 'and / or 3' nucleotide sequences that naturally flank the nucleic acid molecule in the Genomic DNA of the cell from which the nucleic acid comes. In addition, an "isolated" nucleic acid molecule, such as a cDNA molecule, may substantially lack other material cell, or culture medium when produced by techniques recombinants, or substantially lack chemical precursors or other chemicals when chemically synthesized.

As used herein, the expression "hybridizes under conditions of low stringency, medium stringency, high stringency or very high stringency" describes conditions of hybridization and washing. Guidelines for hybridization reactions can be found in Current Protocols in Molecular Biology (1989) John Wiley & Sons, NY, 6.3.1-6.3.6. That reference describes aqueous and non-aqueous methods and both can be used. The specific hybridization conditions referred to herein are the following: 1) low stringency hybridization conditions in sodium chloride / sodium citrate (SSC) 6X at approximately 45 ° C, followed by two washes in 0.2X SSC , 0.1% SDS at least at 50 ° C (the temperature of the washings can be increased to 55 ° C under the conditions of low stringency); 2) hybridization conditions of medium stringency in 6X SSC at approximately 45 ° C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60 ° C; 3) hybridization conditions of high stringency in 6X SSC at approximately 45 ° C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65 ° C; and preferably 4) are hybridization conditions of very high stringency 0.5 M sodium phosphate, 7% SDS at 65 ° C, followed by one or more washes in 0.2X SSC, 1% SDS at 65 ° C. Very high stringency conditions (4) are the preferred conditions and those that should be used unless otherwise specified.

As used herein, a nucleic acid molecule "that is produced in a way natural "refers to an RNA or DNA molecule that has a nucleotide sequence that appears in nature (for example, encodes a natural protein).

As used herein, the "gene" and "recombinant gene" expressions refer to nucleic acid molecules that include a reading phase open encoding a 15603 protein, preferably a 15603 mammalian protein, and may also include sequences non-coding regulators and introns.

An "isolated" polypeptide or protein or "purified" substantially lacks cellular or other material contaminating protein from the cellular or tissue source from which the protein proceeds, or it substantially lacks precursors chemicals or other chemical agents when chemically synthesized. In one embodiment, the expression "substantially lacks" is refers to the preparation of 15603 protein that has less than about 30%, 20%, 10% and more preferably 5% (by weight dry), of protein other than 15603 protein (also called  herein "contaminating protein"), or of chemical precursors or chemical agents that are not protein 15603. When the 15603 protein or the biologically active part of it is produced recombinantly, preferably also lacks substantially culture medium, that is, the medium of crop represents less than about 20%, more preferably less than about 10% and even more preferably less than about 5% of the volume of the protein preparation The invention includes preparations isolated or purified of at least 0.01, 0.1, 1.0 and 10 milligrams in dry weight.

A "non-essential" amino acid residue is a remainder that can be altered from the wild type sequence of 15603  (for example, the sequence of SEQ ID NO: 1 or 3) without canceling or, more preferably, without substantially altering an activity biological, while an "essential" amino acid residue causes such change. For example, it is predictable that the remains amino acids that are conserved among the polypeptides of the present invention, for example, those present in the protein of ionic channel or in the ank repeat domains, whether particularly susceptible to alteration.

A "conservative substitution of amino acid "is one in which the amino acid residue is replaced by an amino acid residue that has a similar side chain. In the technique has defined families of amino acid residues that They have similar side chains. These families include amino acids with basic side chains (for example, lysine, arginine, histidine), acidic side chains (for example, acid aspartic, glutamic acid), unloaded polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β branched side chains (per example, threonine, valine, isoleucine) and side chains aromatic (for example, tyrosine, phenylalanine, tryptophan, histidine). In this way, a non-essential amino acid residue provided in a 15603 protein is preferably replaced by another amino acid residue from the same family of side chains. Alternatively, in another embodiment, they can be introduced random mutations throughout all or part of a coding sequence of 15603, such as by mutagenesis of saturation, and in the resulting mutants the biological activity of 15603 to identify mutants that retain activity. After mutagenesis of SEQ ID NO: 1 or SEC ID No.: 3, the encoded protein can be expressed in a manner recombinant and protein activity can be determined.

As used herein, a "biologically active part" of a 15603 protein includes a fragment of a 15603 protein that participates in an interaction between a 15603 molecule and a different 15603 molecule. The biologically active parts of a 15603 protein they include peptides comprising amino acid sequences sufficiently homologous or derived from the amino acid sequence of the 15603 protein, for example, the amino acid sequence shown in SEQ ID NO: 2, which includes fewer amino acids than the full length 15603 protein, and it has at least one activity of a 15603 protein. Typically, the biologically active parts comprise a domain or motif with at least one activity of the 15603 protein, for example, the ability to regulate the flow of cations through a membrane. A biologically active part of a 15603 protein can be a polypeptide that has, for example, a length of 10, 25, 50, 100, 200 or more amino acids. Biologically active parts of a 15603 protein can be used as targets to develop agents that modulate an activity mediated by 15603, for example, the ability to regulate the flow of cations through the
membrane.

Homology or identity calculations of sequence (the terms "homology" and "identity" are used interchangeably herein) between sequences are Perform as follows:

To determine the percent identity of two amino acid sequences, or two acid sequences nucleic, the sequences are aligned to get a comparison optimal (for example, gaps can be introduced in one or both in the first as in the second amino acid or acid sequences nucleic for optimal alignment and can be discarded non-homologous sequences for comparative purposes). In one embodiment preferred, the length of a reference sequence aligned to comparison purposes is at least 30%, preferably at minus 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at minus 70%, 80%, 90% or 100% of the sequence length of reference (for example, when a second sequence is aligned with the 15603 amino acid sequence of SEQ ID NO: 2 which has 931 amino acid residues, line up at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60% and even more preferably at least 70%, 80% or 90% amino acid residues). Then the amino acid or nucleotide residues at amino acid positions or at the corresponding nucleotide positions. When a position of the first sequence is occupied by the same rest amino acid or nucleotide that the corresponding position in the second sequence, then the molecules are identical in that position (as used herein, "identity" of amino acid or nucleic acid is equivalent to "homology" of amino acid or nucleic acid). The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, that need to be introduced for optimal alignment of the two sequences.

Sequence comparison and determination of percent identity between two sequences can be performed using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 444-453 that has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6. In another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1 , 2, 3, 4, 5 or 6. A particularly preferred series of parameters (and the one that should be used if the specialist doubts which parameters should be applied to determine if a molecule is within a limitation of identity or sequence homology of the invention) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4 and a slot gap penalty phase setting of 5.

The percentage of identity between two sequences of amino acids or nucleotides can be determined using the algorithm of Meyers and Miller ((1989) CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a weight residue table PAM120, a 12-hole gap penalty and one hole penalty of 4.

The nucleic acid and protein sequences described herein can be used as a "search sequence" to perform a search against public databases to identify, for example, other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al . (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to 15603 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to 15603 protein molecules of the invention. To obtain alignments with gaps for comparative purposes, Gapped BLAST can be used as described in Altschul et al ., (1997) Nucleic Acids Res. 25: 3389-3402. When the BLAST and Gapped BLAST programs are used, the default parameters of the respective programs (for example, XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Certain particular 15603 polypeptides have amino acid sequences substantially identical to the sequence of amino acids of SEQ ID NO: 2. In the context of a sequence of amino acids, the expression "substantially identical" is used to refer to a first amino acid that contains a number sufficient or minimum amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and the second amino acid sequences can have a domain common structural and / or a common functional activity. For example, amino acid sequences that contain a structural domain common that has an identity of at least about 60% or 65%, probably a 75% identity, more likely a identity of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 2 they are called substantially identical.

In the context of a nucleotide sequence, the term "substantially identical" is used herein memory to refer to a first acid sequence nucleic that contains a sufficient or minimum number of nucleotides which are identical to nucleotides aligned in a second sequence of nucleic acid such that the first and second sequences of nucleic acid encode a polypeptide that has an activity functional common, or encode a structural polypeptide domain common or a common functional polypeptide activity. For example, sequences and nucleotides that have an identity of at least approximately 60% or 65%, probably an identity of 75%, more probably an identity of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 1 or 3 are called substantially identical.

"Wild wrong or aberrant expression", as used herein, it refers to a pattern of the Gene expression of RNA or non-wild type protein. Includes: expression at levels that are not wild type, is say, excessive or insufficient expression; an expression pattern which differs from the wild type in terms of the moment or phase in the that the gene is expressed, for example, increase or decrease in expression (compared to the wild type) in a period or default development phase; an expression pattern that differs from the wild type in terms of decrease in expression (compared to the wild type) in a type of predetermined cell or tissue type; an expression pattern that differs from the wild type in terms of ayuste size, amino acid sequence, post-translational modification or activity biological of the expressed polypeptide; an expression pattern that differs from the wild type in terms of the effect of a stimulus environmental or extracellular stimulus on gene expression, by example, a pattern of increase or decrease in expression (in comparison with the wild type) in the presence of an increase or decrease in stimulus intensity.

"Subject", as used herein memory, can refer to a mammal, for example, a being human, or to an experimental or animal disease model. He subject can also be a non-human animal, for example, a horse, cow, goat or other pet.

A "purified cell preparation", as used herein, refers, in the case of animal or plant cells, to an in vitro preparation of cells and not to the entire plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject's cells.

Various details are described below. aspect of the invention.

Isolated nucleic acid molecules

This document describes a isolated or purified nucleic acid molecule, which encodes a 15603 polypeptide, for example a 15603 protein in length complete or a fragment thereof, for example, a part biologically active of a 15603 protein. A nucleic acid fragment suitable for use as a probe hybridization that can be used, for example, to identify a nucleic acid molecule encoding a 15603 polypeptide, mRNA of 15603 and fragments suitable for use as primers, for example, PCR primers for amplification or mutation of nucleic acid molecules.

Preferably, a nucleic acid molecule of 15603 isolated includes the nucleotide sequence shown in the SEQ ID NO: 1, or any part of this sequence of nucleotides Preferably, the nucleic acid molecule includes sequences encoding the human 15603 protein (ie, "the coding region "of SEQ ID NO: 1, as shown in the SEQ ID NO: 3). Alternatively, the nucleic acid molecule can  include only the coding region of SEQ ID NO: 1 (for example, SEQ ID NO: 3) and, for example, no flanking sequence of those that normally accompany the object sequence. How alternatively, the nucleic acid molecule encodes a sequence corresponding to a fragment of the protein from approximately amino acid 493 to 727 or from 123 to 347 of SEQ ID NO: 2.

Alternatively, an isolated 15603 nucleic acid molecule includes a nucleic acid molecule that is a complement to the nucleotide sequence shown in SEQ ID NO: 1 or in SEQ ID NO: 3, or a part of any of these nucleotide sequences Alternatively, the nucleic acid molecule is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 such that it can hybridize with the nucleotide sequence shown in SEQ ID NO: 1 or 3, thus forming a duplex
stable.

Alternatively, a nucleic acid molecule of 15603 isolated includes a nucleotide sequence that has a homology of at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater with length integer of the nucleotide sequence shown in SEC No.: 1 or SEC ID No.: 3, or a part, preferably of the same length, of any of those nucleotide sequences.

       \ vskip1.000000 \ baselineskip
    

15603 Nucleic Acid Fragments

A 15603 nucleic acid molecule can include only part of the SEC nucleic acid sequence ID No.: 1 or 3. For example, said nucleic acid molecule can include a fragment that can be used as a probe or primer or a fragment encoding a part of a 15603 protein, for example, an immunogenic or biologically active part of a protein 15603. A fragment may comprise the nucleotides of SEQ ID Nº: 1, which encode an ionic channel protein domain of the 15603 human protein. The nucleotide sequence determined to from the cloning of the 15603 gene allows the generation of probes and primers designed for use in identification and / or cloning of other family members of 15603, or fragments of the same, as well as homologs of the 15603 protein, or fragments of it, coming from other species.

Alternatively, a nucleic acid includes a nucleotide sequence that includes part, or the entire region coding and extends to the 5 'or 3' non-coding region (or both). Other alternatives include a fragment that includes a nucleotide sequence encoding an amino acid fragment described herein. The nucleic acid fragments they can encode a specific domain or site described in the present memory or fragments thereof, particularly fragments thereof with a length of at least 100, 200 or 300 amino acids Fragments also include acid sequences nucleic corresponding to specific amino acid sequences described above or fragments thereof. Should not be considered that nucleic acid fragments include fragments that may have been described before the invention.

A nucleic acid fragment may include a sequence corresponding to a domain, region or functional site described herein. A nucleic acid fragment it can also include one or more domains, regions or sites functionalities described herein. In this way, for example, a 15603 nucleic acid fragment may include a sequence corresponding to an ion channel protein domain, as described herein.

15603 probes and primers are described. Typically, a probe / primer is an isolated oligonucleotide or purified. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under stringent conditions with at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sequence with sense or antisense of SEQ ID NO: 1 or SEQ ID NO: 3, or a natural allelic variant or mutant of SEQ ID NO: 1 or SEQ ID Nº: 3.

In a preferred embodiment, the nucleic acid it is a probe that has a length of at least 5 or 10, and less than 200, more preferably less than 100 or less than 50 base pairs. It must be identical or differ on one or less than 5 or 10 bases from one sequence described herein. If a alignment for this comparison, the sequences must be aligned for maximum homology. The sequences are considered differences of "loops" of deletions or insertions, or decoupling

A probe or primer can come from the sense or antisense chain of a nucleic acid encoding: transmembrane domains of 15603, which are located approximately in amino acid residues 407 to 426, 443 to 459, 490 to 507, 598 to 614, 637 at 653 and 703 to 727 of SEQ ID NO: 2 (provided by MEMSAT, Jones et al . (1994) Biochemistry 33: 3038-3049); or of the Ank repeat domain in amino acids 97 to 131, 132 to 163 and 218 to 250 of SEQ ID NO: 2.

A series of primers is also described, by example, primers suitable for use in a PCR, which can be used to amplify a selected region of a sequence of 15603, for example, a domain, region, site or other sequence described in This memory. The primers must have a length of at minus 5, 10 or 50 base pairs and a length less than 100 or less of 200 base pairs. The primers must be identical or differ on a basis of a sequence described herein or of A natural variant

A nucleic acid fragment can encode an epitope carrying a region of a polypeptide described in the present memory

A nucleic acid fragment encoding a "biologically active part of a 15603 polypeptide" can be prepared by isolating a part of the nucleotide sequence of SEQ ID NO: 1 or 3, which encodes a polypeptide having a biological activity of 15603 (for example the biological activities of the 15603 proteins are described herein), expression of the encoded part of the 15603 protein (for example, by in vitro recombinant expression) and evaluation of the activity of the encoded part of the 15603 protein For example, a nucleic acid fragment encoding a biologically active part of 15603 includes an ion channel domain, for example, amino acid residues approximately 493 to 727 of SEQ ID NO: 2. A nucleic acid fragment encoding a biologically active part of a 15603 polypeptide, may comprise a nucleotide sequence with a length greater than 200, 250, 300 or more nucleotide two.

Preferably, a nucleic acid includes a nucleotide sequence that has a length of approximately 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2100, 2200, 2300, 2400, 2500, 2600, 2700 or more nucleotides and hybridizes under stringent conditions with a nucleic acid molecule of SEQ ID NO: 1 or SEQ ID NO: 3.

       \ vskip1.000000 \ baselineskip
    

15603 Nucleic Acid Variants

Nucleic acid molecules are also described. which differ from the nucleotide sequence shown in SEQ ID Nº: 1 or in SEQ ID NO: 3. These differences may be due to the degeneracy of the genetic code (and produce a nucleic acid that encodes the same 15603 proteins that have been encoded by the nucleotide sequence described herein). How alternatively, an isolated 15603 nucleic acid molecule has a nucleotide sequence that encodes a protein that has a amino acid sequence that differs, at least 1, but less than 5, 10, 20, 50 or 100 amino acid residues shown in the SEC ID No.: 2. If an alignment is needed for this comparison, the sequences must be aligned for maximum homology. They are considered differences the sequences of "loops" of deletions or insertions, or decoupling.

Nucleic acids having codons, which are preferred or non-preferred, can be chosen for a particular expression system. For example, the nucleic acid may be one in which at least one codon, preferably at least 10% or 20% of the codons have been altered so that the sequence is optimized with respect to expression in E. coli, cells of yeast, human cells, insect cells or CHO cells.

The variants of the nucleic acid can be natural, such as allelic variants (same locus), homologous (different locus) and orthologs (different organism) or may be not natural Unnatural variants can be obtained by techniques of mutagenesis, including those applied to polynucleotides, cells or organisms. Variants may contain substitutions, nucleotide deletions, inversions and insertions. May occur a variation in any or both in the region coding as in the non-coding region. Variations they can produce conservative amino acid substitutions and not conservatives (compared to the coded product).

Preferably, the nucleic acid differs from of SEQ ID NO: 1 or 3, for example, as follows: at least one but in less than 10, 20, 30 or 40 nucleotides; to the less in one but in less than 1%, 5%, 10% or 20% of nucleotides in the nucleic acid to analyze. If necessary for this analysis, The sequences must be aligned to achieve maximum homology. The "loop" sequences of deletions or insertions, or decoupling.

Orthologs, counterparts and allelic variants using methods known in the art. These variants comprise a nucleotide sequence that encodes a polypeptide that has an identity of 50%, at least about 55%, typically at least about 70-75%, more typically at least about 80-85% and even more typically at least approximately 90-95% or greater with the sequence of nucleotides shown in SEQ ID NO: 2 or a fragment of this sequence. These nucleic acid molecules can be identified easily as molecules capable of hybridizing under conditions stringent, with the nucleotide sequence shown in SEQ ID Nº: 2 or a fragment of the sequence. Acid molecules nucleic corresponding to orthologs, homologues and variants allelics of the 15603 cDNAs of the invention can be isolated additionally by mapping on the same chromosome or locus as the gene of 15603.

Preferred variants include those that are correlated with the ability to regulate the flow of cations to through a membrane by the presence of a second messenger such as diacylglycerol.

Allelic variants of 15603, for example 15603 human, include both functional proteins and proteins non-functional Functional allelic variants are variants of natural amino acid sequences of the 15603 protein within a population that maintains the ability to regulate channels of cations powered by second messenger activated by diacylglycerol Functional allelic variants typically they will contain only the conservative substitution of one or more amino acids of SEQ ID NO: 2, or the substitution, deletion or insertion of non-critical remains in non-critical regions of the protein. Non-functional allelic variants are variants of natural amino acid sequences of the 15603 protein, by example, the human 15603 protein, within a population that does not they have the ability to regulate cationic channels powered by second messenger activated by diacylglycerol. Variants non-functional allelics will typically contain a substitution, deletion, or non-conservative insertion, or premature truncation of the amino acid sequence of SEQ ID NO: 2, or a substitution, insertion, or deletion in critical debris or regions protein reviews.

In addition, acid molecules are also described. nucleic encoding other family members of 15603 and, by therefore, they have a nucleotide sequence that differs from those 15603 sequences of SEQ ID NO: 1 or SEQ ID NO: 3.

       \ vskip1.000000 \ baselineskip
    

Antisense Nucleic Acid Molecules, Ribozymes and Molecules of Modified 15603 Nucleic Acid

Also described herein is a isolated nucleic acid molecule that is antisense with respect to 15603 protein. An "antisense" nucleic acid can include a nucleotide sequence that is complementary to a "meaningful" nucleic acid encoding a protein, by example, complementary to the coding chain of a molecule of Double stranded or complementary cDNA to an mRNA sequence. Acid antisense nucleic can be complementary to a chain encoder of 15603 integer, or only part of it (for example, the coding region of human 15603 corresponding to the SEQ ID NO: 3). Alternatively, the nucleic acid molecule antisense is antisense with respect to a "region not coding "of the coding chain of a sequence of nucleotides encoding 15603 (for example, regions not translated 5 'and 3').

An antisense nucleic acid can be designed as such that it is complementary to the entire coding region of the MRNA of 15603, but more preferably it is an oligonucleotide that is antisense with respect to only part of the region coding or non-coding of the mRNA of 15603. For example, the antisense oligonucleotide may be complementary to the region surrounding the translation initiation site of the mRNA of 15603, for example, between the -10 and +10 regions of the sequence of nucleotides of the target gene of interest. An oligonucleotide antisense can have, for example, approximately 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more nucleotides of length.

An antisense nucleic acid can be constructed. using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an acid antisense nucleic (for example, an antisense oligonucleotide) it can be chemically synthesized using the nucleotides that are naturally produced or modified nucleotides of very different ways designed to increase biological stability of the molecules or to increase the physical stability of the duplex formed between antisense and sense nucleic acids; by For example, phosphorothioate and nucleotide derivatives can be used with acridine substitutions. Antisense nucleic acid it can also be produced biologically using a vector of expression in which a nucleic acid has been subcloned into a antisense orientation (i.e., RNA transcribed from inserted nucleic acid will have an antisense orientation with with respect to a target nucleic acid of interest, described additionally in the following subsection).

Antisense nucleic acid molecules are typically administered to a subject (for example, by direct injection into a tissue site), or generated in situ such that they hybridize or bind to cellular mRNA and / or genomic DNA encoding a 15603 protein to inhibit protein expression in this way, for example, by inhibiting transcription and / or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then systemically administered. For systemic administration, the antisense molecules can be modified such that they specifically or selectively bind to receptors or antigens expressed on a given cell surface, for example, by binding the antisense nucleic acid molecules to peptides or antibodies that bind to receptors. or cell surface antigens. Antisense nucleic acid molecules can also be administered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

Alternatively, the antisense nucleic acid molecule is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with the complementary RNA in which, unlike the usual β units, the chains circulate in parallel (Gaultier et al . (1987) Nucleic Acids. Res . 15: 6625-6641). The antisense nucleic acid molecule may also comprise a 2'-o-methylribonucleotide (Inoue et al . (1987) Nucleic Acids Res . 15: 6131-6148) or a chimeric RNA-DNA analog (Inoue et al . (1987) FEBS Lett . 215: 327-330).

Alternatively, an antisense nucleic acid is a ribozyme. A ribozyme having specificity for a nucleic acid encoding 15603 may include one or more sequences complementary to the nucleotide sequence of a cDNA of 15603 described herein (ie, SEQ ID NO: 1 or SEQ ID NO: 3 ), and a sequence having a known catalytic sequence responsible for mRNA cleavage (see US Patent No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334: 585-591). For example, a derivative of Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved into a mRNA encoding 15603. See, for example, Cech et al . U.S. Patent No. 4,987,071; and Cech et al . U.S. Patent No. 5,116,742. Alternatively, the 15603 mRNA can be used to select a catalytic RNA that has a specific ribonuclease activity from a group of RNA molecules. See, for example, Bartel and Szostak (1993) Science 261: 1411-1418.

The expression of the 15603 gene can be inhibited by direction to nucleotide sequences complementary to the regulatory region of the 15603 protein (for example, the promoter and / or enhancers of 15603) to form triple helix structures that prevent transcription of the 15603 gene in target cells. See, in general, Helene (1991) Anticancer Drug Des . 6: 569-84; Helene (1992) Ann. NY Acad. Sci . 660: 27-36; and Maher (1992) Bioassays 14: 807-15. Potential sequences that can be set as targets for the formation of triple helices can be increased by creating a so-called "changing"("switchback") nucleic acid molecule. Switchback molecules are synthesized in an alternate 5'-3 ', 3'-5' form, so that they form base pairs first with a chain of one duplex and then with the other, eliminating the need for a considerable stretch of purines or pyrimidines in a duplex chain.

Also described herein are detectably labeled probe molecules and oligonucleotide primers. Typically, these markers are chemiluminescent, fluorescent, radioactive or colorimetric.
Tricos

A nucleic acid molecule of 15603 can be modified in the base moiety, sugar moiety or phosphate backbone to improve, for example, the stability, hybridization or solubility of the molecule. For example, the main deoxyribose phosphate chain of nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup et al . (1996) Bioorganic & Medicinal Chemistry 4: 5-23). As used herein, the terms "nucleic acid peptide" or "APN" refer to a nucleic acid mimetic, for example a DNA mimetic, in which the deoxyribose phosphate backbone is replaced by a backbone. pseudopeptide and only the four natural nucleobases are conserved. The neutral main chain of an APN can allow specific hybridization to DNA and RNA under conditions of high ionic intensity. PNA oligomer synthesis can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al. (1996) supra ; Perry-O'Keefe et al . (1996) Proc. Natl Acad. Sci . 93: 14670-675.

APN of 15603 nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, APN can be used as antisense agents or antigens for the specific modulation of the gene expression sequence, for example, by inducing the stopping of transcription or translation or inhibiting replication. APN of the 15603 nucleic acid molecules can also be used in the analysis of mutations of a single base pair in a gene, (for example, by APN-directed PCR clamping); as "artificial restriction enzymes" when used in combination with other enzymes (eg, S1 nucleases [Hyrup, et al. (1996) supra ]); or as probes or primers for DNA sequencing or hybridization (Hyrup, et al. (1996) supra ; Perry-O'Keefe supra ).

Alternatively, the oligonucleotide may include other pendant groups such as peptides (for example, to target host cell receptors in vivo ), or agents that facilitate transport across the cell membrane (see, for example, Letsinger et al. . (1989) Proc. Natl. Acad. Sci . USA 86: 6553-6556; Lemaitre et al . (1987) Proc. Natl. Acad. Sci . USA 84: 648-652; PCT Publication No. WO 88/09810 ) or the blood brain barrier (see, for example, PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with cleavage agents that are triggered by hybridization (see, for example, Krol et al . (1988) Bio-Techniques 6: 958-976) or intercalating agents. (see, for example, Zon (1988) Pharm. Res . 5: 539-549). For this purpose, the oligonucleotide can be conjugated to another molecule (eg, a peptide, hybridization induced crosslinking agent, transport agent or hybridization induced cleavage agent).

Also described herein are oligonucleotide primer and probe molecules with molecular beacons that have at least one region that is complementary to a 15603 nucleic acid, two complementary regions having one a fluorophore and the other having an inactivator such that the Molecular beacon is useful for quantifying the presence of the 15603 nucleic acid of the invention in a sample. Nucleic acids from molecular beacons are described, for example, in Lizardi et al ., US Patent No. 5,854,033; Nazarenko et al ., U.S. Patent No. 5,866,336 and Livak et al ., U.S. Patent 5,876,930.

       \ vskip1.000000 \ baselineskip
    

Polypeptides 15603 Isolated

Also described herein is a isolated 15603 protein, or a fragment, for example, a part biologically active, for use as immunogens or antigens for induce or test (or more generally to bind) antibodies anti-15603. The 15603 protein can be isolated at from cellular or tissue sources using techniques of Conventional protein purification. The 15603 protein or its fragments can be produced by recombinant DNA techniques or They can be chemically synthesized.

The polypeptides described herein include those that occur as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events and alternative translation and post-translational events. The polypeptide can be expressed in systems, for example, cultured cells that cause substantially the same post-translational modifications present when the polypeptide is expressed in a native cell, or in systems that cause the alteration or omission of post-translational modifications, for example glycosylation or cleavage, present in a cell
native

Preferably, a 15603 polypeptide has a or more of the following features:

It has the ability to regulate the flow of cations through a membrane, the ability to bind to a second messenger, the ability to join diacylglycerol, the ability to regulate intracellular calcium levels and the ability to regulate angiogenesis;

it has a molecular weight, for example, a weight molecular deduction, preferably ignoring any contribution  of post-translational modifications, amino acid composition or other physical characteristics of a 15603 polypeptide, for example a polypeptide of SEQ ID NO: 2;

has a global sequence similarity of to minus 60%, preferably at least 70%, more preferably at minus 80, 90 or 95%, with a polypeptide of SEQ ID NO: 2;

it is expressed in at least the following human tissues and cell lines: at high levels in names of tissues in the list and taqman: hemangioma, vascular smooth muscle, megakaryocytes, Wilm tumor; at medium levels in normal cerebral cortex, uterus and proliferative endothelial cells; and at low levels in adipose tissue, normal human internal mammary artery, diseased human aorta, diseased human artery, diseased human vein and normal human vein, normal heart, CHF heart, normal kidney, skeletal muscle, pancreas, primary osteoblasts, cerebral hypothalamus, breast normal, normal ovary, normal prostate, prostate tumor, salivary glands, normal colon, colon tumor, normal lung, lung tumor, pulmonary COPD, colon IBD, normal spleen, normal tonsil, normal small intestine, pressure ulcer in skin, synovium, glioblastomas, fetal adrenal gland, fetal liver and heart
fetal;

has an ionic channel protein domain that it has an identity preferably of about 70%, 80%, 90% or 95% to amino acid residues approximately 493 to 727 of the SEQ ID NO: 2;

has an ank repeat domain that has an identity of preferably about 70%, 80%, 90% or 95% with the amino acid residues about 97 to 131, 132 to 163 and 218 to 250 of SEQ ID NO: 2;
Y

Preferably, the 15603 protein, or its fragment, differs from the corresponding sequence of SEQ ID NO: 2. In one embodiment it differs at least by one but less than 15, 10 or 5 amino acid residues. In another it differs from the sequence corresponding to SEQ ID NO: 2 in at least one remainder but less of 20%, 15%, 10% or 5% of the remains in it differ from the corresponding sequence of SEQ ID NO: 2. (If this comparison requires an alignment of the sequences, must be aligned to maximum homology The sequences of "loops" due to deletions or insertions, or decoupling, are considered differences). The differences are preferably differences or changes in a non-essential rest or a conservative substitution. Preferably, the differences are not in the domain of repetition of ank approximately in the remains 97 to 131, 132 to 163 and 218 to 250 of SEQ ID NO: 2. Alternatively, one or more differences are in the ank repeat domain approximately  in residues 97 to 131, 132 to 163 and 218 to 250 of SEQ ID NO: 2.

Other alternatives include a protein that contains one or more changes in the amino acid sequence, for for example, a change in an amino acid residue that is not essential for activity. These 15603 proteins differ in the sequence of amino acids of SEQ ID NO: 2, although they retain activity biological

Alternatively, the protein includes a amino acid sequence with a homology of at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or greater with SEQ ID NO: 2.

A 15603 protein or fragment is provided which varies from the sequence of SEQ ID NO: 2 in defined regions by about amino acid residues 407 to 426, 443 to 459, 490 to 507, 598 to 614, 637 to 653, or 703 to 727 of SEQ ID NO: 2 to less in one but less than 15, 10 or 5 amino acid residues of the protein or fragment In some alternatives, the difference is in a non-essential rest or is a conservative substitution, while that in others the difference is in an essential rest or is a non-conservative substitution

A 15603 protein or fragment is provided which varies from the sequence of SEQ ID NO: 2 in defined regions by amino acid residues approximately 97 to 131, 132 to 163 and 218 to 250 of SEQ ID NO: 2 in at least one but less than 15, 10 or 5 amino acid residues in the protein or fragment. In some alternatives, the difference is in a non-essential rest or is a conservative substitution, while in others the difference is in an essential residue or is a non-conservative substitution.

In an alternative, a biologically part active of a 15603 protein includes at least one domain transmembrane In addition, other parts can be prepared biologically active in which other regions are deleted of the protein by recombinant techniques and evaluated with respect to one or more of the functional activities of a 15603 protein native

Preferably, the 15603 protein has a amino acid sequence shown in SEQ ID NO: 2. In others embodiments, the 15603 protein is sufficiently or substantially identical to SEQ ID NO: 2. Alternatively, the 15603 protein is sufficiently or substantially identical to the SEQ ID NO: 2 and retains the functional activity of the protein in the SEQ ID NO: 2, as described in detail in the subsections previous.

       \ vskip1.000000 \ baselineskip
    

15603 Chimeric or Fusion Proteins

Also described herein chimeric or fusion proteins of 15603. As used in the present memory, a "chimeric protein" or "protein of fusion "of 15603 includes a 15603 polypeptide bound to a polypeptide that is not 15603. A "polypeptide that is not 15603" refers to a polypeptide that has an amino acid sequence corresponding to a protein that is not substantially homologous to 15603 protein, for example, a protein that is different from the 15603 protein and that comes from the same organism or an organism different. Fusion protein 15603 polypeptide can correspond to all or a part, for example, to a fragment described herein of an amino acid sequence of 15603. Preferably, a fusion protein of 15603 includes the least one (or two) biologically active parts of a protein 15603. Polypeptide that is not 15603 can be fused to the N-terminus or to the C-terminus of polypeptide 15603.

The fusion protein may include a moiety that It has high affinity for a ligand. For example, the protein of fusion can be a fusion protein GST-15603 in which the sequences of 15603 are fused to the C end of the GST sequences. These fusion proteins can facilitate the Purification of recombinant 15603. As an alternative, the protein fusion can be a 15603 protein that contains a sequence heterologous signal at its N end. In certain host cells (e.g., mammalian host cells), expression and / or 15603 secretion can be increased by the use of a heterologous signal sequence.

Fusion proteins can include all or a part of a serum protein, for example a part of a immunoglobulin (for example IgG, IgA, or IgE), for example a Fc region and / or the hinge sequences C1 and C2 of a Human serum immunoglobulin or albumin.

The 15603 fusion proteins can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The 15603 fusion proteins can be used to affect the bioavailability of a 15603 substrate. The 15603 fusion proteins can be therapeutically useful for the treatment of disorders produced, for example, by i) aberrant mutation or modification of a gene that encodes a 15603 protein; ii) the altered regulation of the 15603 gene; and (iii) an aberrant post-translational modification of a 15603 protein.

In addition, 15603 fusion proteins can be used as immunogens to produce antibodies anti-15603 in a subject, to purify ligands from 15603 and in screening assays to identify molecules that inhibit the interaction of 15603 with a substrate of 15603.

It is available in the vector market of expression that already encodes a fusion remainder (for example, a GST polypeptide). A nucleic acid encoding 15603 can cloned into said expression vector so that the rest of fusion binds to 15603 protein.

       \ vskip1.000000 \ baselineskip
    

15603 Protein Variants

Also described herein is a variant of a 15603 polypeptide, for example, that functions as a agonist (mimetic) or as an antagonist. Can be generated 15603 protein variants by mutagenesis, for example by discrete point mutation, insertion or deletion of sequences or truncation of a protein 15603. a protein agonist 15603 may retain substantially the same, or a subset of the biological activities of the natural form of a protein 15603. An antagonist of a 15603 protein may inhibit one or more of the activities of the natural form of the 15603 protein, by for example, by competitive modulation of an activity mediated by 15603 of a 15603 protein. Therefore, they can be triggered specific biological effects by treatment with a limited function variant. Preferably, the treatment of a subject with a variant that has a subset of activities Biological of the natural form of the protein has less effects side effects in a subject with respect to treatment with the form Natural protein 15603.

Variants of a protein can be identified 15603 exploring in combinatorial libraries of mutants, by example, truncation mutants, of a 15603 protein the agonist or antagonistic activity.

Fragment libraries can be used, for eg, N-terminal, C-terminal, or internal fragments of a 15603 protein coding sequence to generate a population Varied fragments for exploration and subsequent selection of variants of a 15603 protein.

Variations are particularly preferred in the that a cysteine residue is added or deleted or in which add or delete a residue that is glycosylated.

Methods for exploring gene products from combinatorial libraries obtained by point mutations or truncation, and for scanning cDNA libraries to select gene products having a selected property are known in the art. Joint recursive mutagenesis (REM), a new technique that improves the frequency of functional mutants in libraries, can be used in combination with screening tests to identify variants of the 15603 protein (Arkin and Yourvan ( 1992) Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave et al . (1993) Protein Engineering 6: 327-331).

Cell-based assays can be exploited to analyze a library of 15603 varied. For example, an expression vector library can be transfected into a cell line, for example, a cell line that normally responds to 15603 in a substrate dependent manner. Transfected cells are then contacted with 15603 and the effect of mutant expression on signaling by substrate 15603 can be detected, for example by measuring membrane potentials or binding to diacylglycerol. The plasmid DNA can then be recovered from the cells that reflect the inhibition or, alternatively, the potentiation of the signaling by the 15603 protein substrate, and further characterize the different
clones

Also described herein is a method for obtaining a 15603 polypeptide, for example a peptide which has an activity of the wild type, for example, a antagonist, agonist or superagonist of a 15603 polypeptide natural, for example a natural 15603 polypeptide. The method includes alter the sequence of a 15603 polypeptide, for example, alter the sequence, for example, by substitution or deletion of one or more remains of a non-conserved region, a domain or remainder described in the present report, and test on the altered polypeptide the desired activity

Also described herein is a method to obtain a fragment or analogue of a 15603 polypeptide with a biological activity of a natural 15603 polypeptide. He method includes altering the sequence, for example, by substitution or deletion of one or more residues of a 15603 polypeptide from, by example, altering the sequence of a non-conserved region, or a domain or remainder described herein, and test in the polypeptide altered the desired activity.

       \ vskip1.000000 \ baselineskip
    

Anti-15603 antibodies

Also described herein is a anti-15603 antibody. The term "antibody" as used herein, refers to a molecule immunoglobulin or an immunologically active part thereof, that is, an antigen binding part. The parts examples Immunologically active immunoglobulin molecules include scFV and dcFV fragments, and Fab and F (ab ') 2 fragments that they can be generated by treating the antibody with such an enzyme as papain or pepsin, respectively.

The antibody can be an antibody polyclonal, monoclonal, recombinant, for example, chimeric or humanized, completely human, nonhuman, for example, murine, or single chain In a preferred embodiment it has a function effector and can be fixed to the complement. The antibody can coupled to a toxin or an imaging agent.

A 15603 full length protein or a 15603 antigenic peptide fragment can be used as an immunogen  or can be used to identify antibodies anti-15603 obtained with other immunogens, by example, cells, membrane preparations and the like. Peptide 15603 antigen must include at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 and includes a epitope of 15603. Preferably, the antigenic peptide includes the at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues and even more preferably at least 30 residues amino acids.

       \ vskip1.000000 \ baselineskip
    

Recombinant Expression Vectors, Hosting Cells and Cells Obtained By Genetic Engineering

They are also described herein. vectors, preferably expression vectors containing a nucleic acid encoding a polypeptide described herein memory. As used herein, the term "vector" refers to a nucleic acid molecule capable of transport another nucleic acid to which it has bound and may include a plasmid, cosmid or viral vector. The vector may be able to replicate autonomously or can be integrated into the DNA of a host. Viral vectors include, for example, retroviruses. with defects in replication, adenovirus and virus adenoasociados.

A vector may include a nucleic acid of 15603 in a form suitable for nucleic acid expression in a host cell Preferably, the expression vector recombinant includes one or more linked regulatory sequences operatively to the nucleic acid sequence to be expressed. He term "regulatory sequence" includes promoters, enhancers and other expression control elements (for example polyadenylation signals). Regulatory sequences include those that direct the constitutive expression of a sequence of nucleotides, as well as inducible and / or regulatory sequences with tissue specificity The expression vector design can depend on factors such as the choice of the host cell to be transformed, the desired protein expression level and Similar. Expression vectors can be introduced into cells hosts to produce proteins or polypeptides in this way, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (by example, 15603 proteins, 15603 protein mutant forms, fusion proteins and the like).

Recombinant expression vectors can be designed for the expression of 15603 proteins in prokaryotic or eukaryotic cells. For example, polypeptides of the invention can be expressed in E. coli , insect cells (for example using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are further described in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 1885, Academic Press, San Diego, CA. Alternatively, the recombinant expression vector can be transcribed and translated in vitro , for example, using T7 promoter regulatory sequences and T7 polymerase.

Protein expression in prokaryotes is most often performed in E. coli with vectors containing constitutive or inducible promoters that direct the expression of fusion proteins or proteins that are not fusion proteins. Fusion vectors add several amino acids to a protein encoded therein, usually at the amino terminus of the recombinant protein. These fusion vectors typically have three purposes: 1) increase the expression of the recombinant protein; 2) increase the solubility of the recombinant protein; and 3) assist in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to allow separation of the recombinant protein from the fusion moiety after purification of the fusion protein. These enzymes and their related recognition sequences include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) that fuse glutathione S-transferase (GST), maltose E binding protein or protein A, respectively, to the target recombinant protein.

Purified fusion proteins can be used in activity tests of 15603 (for example, trials direct or competitive trials described in more detail below), or to generate specific or selective antibodies to 15603 proteins. Preferably, an expressed fusion protein in a retroviral vector it can be used to infect cells of bone marrow that is subsequently transplanted into receptors irradiated Subsequently, the pathology of the recipient is examined after sufficient time has elapsed (for example, six weeks).

To maximize the expression of recombinant proteins in E. coli , the protein is expressed in a host bacterium with an impaired ability to proteolytically cleave the recombinant protein (Gottesman (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those used preferably in E. coli (Wada et al ., (1992) Nucleic Acids Res . 20 : 2111-2118). This alteration of nucleic acid sequences of the invention can be performed by conventional DNA synthesis techniques.

The expression vector of 15603 can be a yeast expression vector, a vector for expression in insect cells, for example, an expression vector of baculovirus or a vector suitable for expression in cells of mammal.

When used in mammalian cells, the expression vector control functions are often provided by viral regulatory elements. For example, the Commonly used promoters come from polyoma, Adenovirus 2, Cytomegalovirus and simian virus 40.

Alternatively, the recombinant mammalian expression vector can direct the expression of the nucleic acid preferably in a particular cell type (for example, tissue specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue specificity promoters include the albumin promoter (with liver specificity; Pinkert et al . (1987) Genes Dev . 1: 265-277), lymphoid specificity promoters (Calame and Eaton (1988) Adv. Immunol. 43: 235-275), in particular T cell receptor promoters (Winoto and Baltimore (1989) EMBO J. 8: 729-733) and immunoglobulins (Banerji et al . (1983) Cell 33: 729- 740; Queen and Baltimore (1983) Cell 33: 741-748), specific neuron promoters (eg, the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86: 5473-5477) , promoters with specificity of the pancreas (Edlund et al . (1985) Science 230: 912-916), and promoters with specificity of the mammary gland (for example, whey promoter; U.S. Patent No. 4,873,316 and Publication of European Application No. 264,166). Also included are regulated promoters in development, for example, murine hox promoters (Kessel and Gruss (1990) Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3 : 537-546).

Also described is a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. Regulatory sequences (eg, viral promoters and / or enhancers) may be chosen operatively linked to a cloned nucleic acid in the antisense orientation that directs the constitutive, tissue-specific or cell-specific expression of the antisense RNA in a variety of cell types. The antisense expression vector may be in the form of a recombinant plasmid, phagemic or attenuated virus. For analysis of gene expression regulation using antisense genes, see Weintraub et al ., (1986) Reviews - Trends in Genetics 1: 1.

Also described herein is a host cell that includes a nucleic acid molecule described herein, for example, an acid molecule 15603 nucleic within a recombinant expression vector or a 15603 nucleic acid molecule that contains sequences that allow it to recombine homologously at a specific site of the genome of the host cell. The expressions "cell host "and" recombinant host cell "are used interchangeably herein. These terms refer not only to the particular object cell, but to the offspring or possible offspring of said cell. Since they can occur certain modifications in successive generations due to mutations or environmental influences, such offspring not it may, in fact, be identical to the original cell, but it follows including within the scope of the terminology as it is used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, a 15603 protein can be expressed in bacterial cells such as E. coli cells, insect cells, yeast cells or mammalian cells (such as Chinese hamster ovary (CHO) cells or the origin of CV-1 or cells SV-40 (COS)). Those skilled in the art know other suitable host cells.

Vector DNA can be introduced into cells hosts by transformation or transfection techniques conventional. As used herein, the terminology "transformation" and "transfection" are intended refer to a series of techniques recognized in the prior art to introduce a foreign nucleic acid (for example, DNA) into a host cell, which include coprecipitation with phosphate from calcium or calcium chloride, transfection by DEAE-dextran, lipofection or electroporation

A host cell can be used to produce (ie express) a 15603 protein. Therefore, the invention further provides methods for producing a protein 15603 using the host cells of the invention. For example, the method includes culturing the host cell of the invention (in which has been introduced a recombinant expression vector that encodes a 15603) protein in a suitable medium such that a 15603 protein is produced. Alternatively, the method includes also isolate a 15603 protein from the medium or cell host

A cell or preparation is also described. purified from cells that include a 15603 transgene, or that erroneously express 15603 for another reason. The preparation of cells can consist of human or non-human cells, by eg rodent cells, for example, mouse or rat cells, rabbit cells or pig cells. Preferably, the cell or cells include a 15603 transgene, for example, a form heterologous to a 15603 protein, for example, a gene from human beings (in the case of a non-human cell). The transgene of 15603 can be expressed defectively, for example, having a Excessive or insufficient expression. Preferably, the cell or cells include a gene that erroneously expresses a protein Endogenous 15603, for example, a gene whose expression is altered, for example, a knockout gene. These cells can serve as model to study disorders that are related to alleles of 15603 mutated or expressed erroneously or for use in the drug selection.

A human cell is also described, by example a hematopoietic stem cell, transformed with an acid nucleic encoding an object 15603 polypeptide.

Also described are cells, preferably human cells, for example, human hematopoietic cells or fibroblasts, in which an endogenous 15603 gene is under the control of a regulatory sequence that normally does not control the expression of the endogenous 15603 gene. The expression characteristics of an endogenous gene within a cell, for example, a cell line or microorganism, can be modified by inserting a regulatory element of heterologous DNA into the genome of the cell such that the inserted regulatory element is operatively linked to the gene. of endogenous 15603. For example, an endogenous 15603 gene that is "transcriptionally silent", for example, is not normally expressed or expressed only at very low levels, can be activated by the insertion of a regulatory element that is capable of promoting the expression of a product gene normally expressed in that cell. Techniques such as homologous recombinations directed to insert heterologous DNA can be used as described, for example, in Chappel, US 5,272,071; WO 91/06667, published May 16,
1991

       \ vskip1.000000 \ baselineskip
    

Applications

Nucleic acid molecules, proteins, protein and antibody homologs described herein  they can be used in one or more of the following methods: a) exploration;

Isolated nucleic acid molecules described herein can be used, for example, to express a 15603 protein (for example, through a vector of recombinant expression in a host cell in applications of gene therapy), to detect an mRNA of 15603 (for example, in a biological sample) or a genetic alteration in a gene of 15603, and to modulate the activity of 15603, as described with more detail later. 15603 proteins can be used to treat disorders characterized by insufficient production or excessive of a 15603 substrate or the production of inhibitors of 15603. In addition, 15603 proteins can be used to explore 15603 natural substrates, to select drugs or compounds that modulate the activity of 15603, as well as to treat disorders characterized by excessive or insufficient production of the 15603 protein or the production of 15603 protein forms that have a reduced, aberrant or unwanted activity in comparison with the wild type 15603 protein (for example, a function or aberrant or deficient expression of the ionic channel). In addition, the anti-15603 antibodies of the invention can used to detect and isolate 15603 proteins, regulate the bioavailability of 15603 proteins and modulate the activity of 15603

A method is provided to evaluate in a compound the ability to interact, for example, to bind to an object 15603 polypeptide. The method includes: contacting the compound with the subject polypeptide 15603; and assess the ability of the compound to interact with, for example, to bind or complex with the object polypeptide 15603. This method can be performed in vitro , for example in a cellless system, or in vivo , for example, in a double hybrid interaction TRAP assay. This method can be used to identify natural molecules that interact with the object 15603 polypeptide. It can also be used to find natural or synthetic inhibitors of the target 15603 polypeptide. Further exploration methods are described in more detail.

       \ vskip1.000000 \ baselineskip
    

Screening tests

The invention provides methods (also referred to herein as "screening tests") to identify modulators, i.e. compounds or agents Candidates or assays (for example, proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which have an inhibitory effect on the expression of 15603 or the 15603 activity. Compounds identified in this way can be used to modulate the activity of target gene products (for example genes of 15603) in a therapeutic protocol, for develop the biological function of the target gene product, or to identify compounds that alter normal interactions with The target gene

Trials to explore are also described candidate or test compounds that are substrates of a protein or 15603 polypeptide or a biologically active part thereof. Assays are also described to explore candidate compounds or assays that bind or modulate the activity of a protein or 15603 polypeptide or a biologically active part of the same.

The test compounds used in the present invention can be obtained using any of the numerous strategies in combinatorial library methods known in the art, including: biological libraries; Peptoid libraries (libraries of molecules that have peptide functionalities, but with a new non-peptide skeleton that are resistant to enzymatic degradation but remain bioactive; see, for example, Zuckermann et al . (1994) J. Med. Chem . 37: 2678-85); libraries in solution phase or in solid phase parallel spatially manageable; synthetic library methods that require deconvolution; the library method "a pearl-a compound"; and synthetic library methods that use affinity chromatography selection. The biological library and peptoid library strategies are limited to peptide libraries, while the other four strategies are applicable to peptide libraries, non-peptide oligomers or small molecule compounds (Lam (1997) Anticancer Drug Des . 12: 145) .

Examples of methods for the synthesis of molecular libraries can be found in the art, for example, in: DeWitt et al . (1993) Proc. Natl Acad. Sci . USA 90: 6909-13; Erb et al . (1994) Proc. Natl Acad. Sci . USA 91: 11422-426; Zuckermann et al . 1994. J. Med. Chem. 37: 2678-85; Cho et al . (1993) Science 261: 1303; Carrell et al . (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl . 33: 2061; and in Gallop et al . (1994) J. Med. Chem. 37: 1233-51.

Compound libraries can be presented in solution (for example, Houghten (1992) Biotechniques 13: 412-421), or in beads (Lam (1991) Nature 354: 82-84), chips (Fodor (1993) Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, U.S. Patent No. '409), plasmids (Cull et al . (1992) Proc Natl Acad Sci USA 89: 1865-1869) or phage (Scott and Smith (1990) Science 249: 386-390; Devlin (1990) Science 249: 404-406; Cwirla et al . (1990) Proc. Natl. Acad. Sci . 87: 6378-6382; Felici (1991) J. Mol Biol 222: 301-310; Ladner supra.)...

In one embodiment, the test is an essay. cell-based in which a cell that expresses a protein 15603 or a biologically active part of it is put into contact with a test compound and the capacity of the test compound inhibiting the activity of 15603. The determination of the ability of the test compound to modulate 15603 activity can be performed by monitoring the function of the ionic channel The cell, for example, can be mammalian, for example, human.

       \ newpage
    

The capacity of the test compound modulating the binding of 15603 to a compound, for example, a substrate of 15603, or joining 15603. This can achieved, for example, by coupling the compound, by example the substrate, with a radioisotopic or enzymatic marker of such that the binding of the compound, for example, the substrate to 15603 can be determined by detecting the compound labeled, for example, the substrate in a complex. As an alternative, 15603 could be coupled with a radioisotopic or enzymatic marker to control the ability of a test compound to modulate the binding of 15603 to a substrate of 15603 in a complex. For example, compounds can be labeled (for example, substrates of 15603) with 121 I, 35 S, 14 C, or 3 H, directly or indirectly, and detect the radioisotope by directly counting radio broadcasting or counting the scintillation. As an alternative, compounds can be labeled enzymatically, for example, with Horseradish peroxidase, alkaline phosphatase, or luciferase, and the Enzymatic marker can be detected by determining the conversion of an appropriate substrate in product.

The ability of a compound (for example, a 15603 substrate) to interact with 15603 with or without the labeling of any of the interacting agents can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with 15603 without labeling the compound or 15603. Protein McConnell et al . (1992) Science 257: 1906-1912. As used herein, a "microphysiometer" (for example, Cytosensor) is an analytical instrument that measures with a light-activated potentiometric sensor (LAPS) the rate at which a cell acidifies its environment. Changes in this acidification rate can be used as an indicator of the interaction between a compound and 15603.

In yet another embodiment, a cell-free assay in which a 15603 protein is contacted with a test compound and the capacity of the compound is evaluated 15603 protein inhibit assay. Biologically parts active ingredients of 15603 proteins to be used in assays described in the This report includes fragments that participate in interactions with molecules that are not 15603, for example, fragments with high probability scores in surface.

In the cellless assays of the invention soluble and / or membrane bound protein forms can be used 15603. When bound forms are used in the protein membrane, It may be desirable to use a solubilizing agent. The Examples of these solubilizing agents include non-detergents. ionics such as n-octylglucoside, n-dodecylglucoside, n-dodecylltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, isotridecipoli (ethylene glycol ether) n, 3 - [(3-colamidopropyl) dimethylammonium] -1-propane  sulphonate (CHAPS), 3 - [(3-colamidopropyl) dimethylammonium] -2-hydroxy-1-propane sulphonate (CHAPSO) or N-dodecyl-N, N-dimethyl-3-ammonium-1-propane  sulphonate

Cellless assays involve preparing a ionic channel in a plasma membrane and measure the flow of ions to through the pore of the membrane, for example by zonal clamping. If ion channel or protein or subunit binding is required related and an activation compound or protein for activation of the channel, this can be achieved in conditions and during a enough time to allow the two components interact and unite, thus forming a complex.

The interaction between two molecules can also be detected, for example, using fluorescence energy transfer (FET) (see, for example, Lakowicz et al ., U.S. Patent No. 5,631,169; Stavrianopoulos, et al ., U.S. Pat. United No. 4,868,103). A fluorophore marker in the first "donor" molecule is selected such that its emitted fluorescent energy is absorbed by a fluorescent marker in a second "acceptor" molecule, which in turn can emit fluorescence due to absorbed energy. Alternatively, the "donor" protein can simply use the natural fluorescent energy of tryptophan residues. Markers that emit different wavelengths of light are chosen, so that the marker of the "acceptor" molecule can be distinguished from that of the "donor." Since the efficiency of energy transfer between the markers is related to the distance between the molecules, the spatial relationship between the molecules can be evaluated. In a situation where the union between the molecules occurs, the fluorescent emission of the "acceptor" molecule marker in the assay must be maximum. A FET binding event can conveniently be measured by conventional fluorometric detection means well known in the art (for example, using a fluorimeter).

Alternatively, the ability of protein 15603 to bind to a target molecule can be determined using real-time biomolecular interaction analysis (BIA) (see, for example, Sjolander and Urbaniczky (1991) Anal. Chem . 63: 2338-2345 and Szabo et al . (1995) Curr. Opin. Struct. Biol. 5: 699-705). "Surface plasmon resonance" or "BIA" detects biospecific interactions in real time, without marking any of the interacting molecules (for example, BIAcore). Changes in the mass in the bonding surface (indicating a bonding event) produce alterations in the index of light refraction near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

For example, the target gene product or the Test substance is anchored in a solid phase. At the end of the reaction gene product complexes can be detected target / test compound anchored in the solid phase. Preferably, the target gene product can be anchored on a solid surface and the test compound (which is not anchored) can be labeled, directly or indirectly, with detectable markers described In the present memory.

       \ newpage
    

It may be desirable to immobilize 15603, or an anti-15603 antibody or its target molecule to facilitate separation between complexed and uncomplexed forms of one or both proteins, as well as to accommodate assay automation. The binding of a test compound to a 15603 protein, or the interaction of a 15603 protein with a target molecule in the presence and absence of a candidate compound, can be performed in any suitable container to contain the reagents. Examples of these containers include microtiter plates, test tubes and microcentrifuge tubes. For example, a fusion protein can be provided that adds a domain that allows one or both proteins to be bound to a matrix. For example, glutathione-S-transferase / 15603 fusion proteins or glutathione-S-transferase / target fusion proteins can be adsorbed on glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione-modified microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed target protein or 15603 protein, and the mixture can be incubated under conditions that lead to complex formation (for example, under physiological conditions for salts and pH) . After incubation, the beads or wells of the microtiter plate are washed to remove any unbound component, the matrix is immobilized in the case of the beads and the complexes are determined directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix and the level of binding or activity of 15603 can be determined using techniques
conventional.

Other techniques to immobilize a protein 15603 or a target molecule in matrices include the use of conjugation of biotin and streptavidin. Molecules can be prepared target or biotinylated 15603 proteins from biotin-NHS (N-hydroxy-succinimide) using techniques known in this field (for example, a kit of biotinylation, Pierce Chemicals, Rockford, IL), and immobilize in the wells of 96-well plates coated with streptavidin  (Pierce Chemical).

To perform the test, the component does not Fixed assets are added to the coated surface that contains the anchored component. After the reaction is complete, they are removed components that have not reacted (for example by washing) in conditions such that the complexes formed remain immobilized on the solid surface. Complex detection anchored on the solid surface can be done in several ways. When the previously unmobilized component is premarked, surface immobilized marker detection indicates that complexes formed. When the non-immobilized component previously not premarked, an indirect marker can be used to detect surface anchored complexes; for example, using a specific or selective labeled antibody for the immobilized component (the antibody, in turn, can be labeled directly or indirectly marked with, for example, a labeled anti-Ig antibody).

For example, this test is performed using antibodies reactive with 15603 protein or target molecules but that does not interfere with the binding of 15603 protein to its molecule Diana. These antibodies can be modified in the wells of the plate and unbound target or 15603 protein can get trapped in the wells by conjugation with the antibody. The methods for detect these complexes, in addition to those described above for complexes immobilized with GST, they include immunodetection of complexes using antibodies reactive with 15603 protein or target molecule, as well as enzyme linked assays that are based on the detection of an enzymatic activity associated with the protein 15603 or target molecule.

Alternatively, cell-free assays can be performed in a liquid phase. In said test, the reaction products are separated from the components that have not reacted, by any of several conventional techniques, including but not limited to: differential centrifugation (see, for example, Rivas and Minton (1993) Trends Biochem Sci 18: 284 -7); chromatography (molecular exclusion chromatography, ion exchange chromatography); electrophoresis (see, for example, Ausubel et al ., eds. (1999) Current Protocols in Molecular Biology , J. Wiley, New York.); and immunoprecipitation (see, for example, Ausubel et al ., eds. (1999) Current Protocols in Molecular Biology , J. Wiley, New York). These resins and chromatographic techniques are known to a person skilled in the art (see, for example, Heegaard (1998) J Mol Recognit 11: 141-8; Hage and Tweed (1997) J Chromatogr B Biomed Sci Appl. 699: 499-525 ). In addition, fluorescence energy transfer, as described herein, can also be conveniently used to detect binding without further purification of the complex of the
solution.

Preferably, the assay includes putting in 15603 protein contact or a biologically active part of the same with a known compound that binds to 15603 to form a test mixture, contact the test mixture with a test compound and determine the ability of the compound to interaction assay with a 15603 protein, where the determination of the capacity of the test compound of interacting with a 15603 protein includes determining the ability of the test compound to preferentially bind 15603 or to a biologically active part of it or modulate the activity of a target molecule compared to the compound known.

The products of the target genes described herein may interact, in vivo, with one or more cellular or extracellular macromolecules, such as proteins. For the purposes of this analysis, these cellular and extracellular macromolecules are referred to herein as "binding partners." Compounds that break said interactions may be useful for regulating the activity of the target gene product. These compounds may include, but are not limited to, molecules such as antibodies, peptides and small molecules. Preferred target products / genes are the 15603 genes identified herein. Also described herein are methods for determining the ability of the test compound to modulate the activity of a 15603 protein by modulating the activity of an effector downstream of a target molecule. For example, the activity of the effector molecule on an appropriate target can be determined or the binding of the effector to an appropriate target can be determined, as previously described.

To identify compounds that interfere with the interaction between the target gene product and its cell or extracellular binding partner, a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a period of time sufficient to allow the two products to form a complex. To test an inhibitory agent, the reaction mixture is provided in the presence and absence of the test compound. The test compound may initially be included in the reaction mixture or may be added within a period of time after the addition of the target gene and its cell or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complex between the target gene product and the cell or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene product and the interactive binding partner. In addition, the formation of complexes within reaction mixtures containing the test compound and the normal target gene product can also be compared with the formation of complexes within reaction mixtures containing the test compound and the mutant target gene product. This comparison may be important in cases where it is desirable to identify compounds that alter mutant interactions but not target gene products.
normal.

These tests can be performed in a format heterogeneous or homogeneous. Heterogeneous trials involve the anchoring of the target gene product or the binding partner in a solid phase and the detection of complexes anchored in the solid phase at the end of the reaction. In homogeneous tests, the reaction Whole is done in a liquid phase. In any strategy, the order of addition of reagents can be varied to obtain different information on the compounds being tested. For example, test compounds can be identified that interfere with the interaction between the target gene products and union partners, for example, by competition performing the reaction in the presence of the test substance. As an alternative, test compounds that break the complexes can be tested preformed, for example, compounds with higher constants of union that displace one of the components of the complex, adding the test compound to the reaction mixture after it is They have formed the complexes. The various formats are described. briefly later.

In a heterogeneous test system, the product target gene or cell or extracellular binding partner interactive, it is anchored on a solid surface (for example, a microtiter plate), while the non-anchored species is brand directly or indirectly. Anchored species can immobilize by covalent or non-covalent junctions. How alternatively, a specific immobilized antibody or selective for the species to anchor to anchor the species to the solid surface

To perform the essay, the partner of the immobilized species is exposed to the surface coated with or without the test compound. After completion of the reaction, it remove unreacted components (for example by washing) and any complex formed will remain immobilized in the solid surface When the non-immobilized species is pre-marked, immobilized marker detection on the surface it indicates that complexes were formed. When the non-immobilized species is not pre-marked, may an indirect marker be used to detect complexes anchored in the surface; for example, using a specific labeled antibody or selective for the species not initially immobilized (the antibody, in turn, can be directly labeled or labeled indirectly with, for example, an antibody marked anti-Ig). Depending on the order of addition of the reaction components, compounds of assay that inhibit complex formation or break the preformed complexes.

Alternatively, the reaction can be carried out in a liquid phase in the presence or absence of the test compound, the reaction products can be separated from the unreacted components and the complexes can be detected; for example, using a specific or selective immobilized antibody for one of the binding components to anchor any complex formed in solution, and a specific or selective labeled antibody for the other partner to detect anchored complexes. Again, depending on the order of addition of the reagents to the liquid phase, test compounds that inhibit complexes or break complexes can be identified
preformed

Alternatively, a homogeneous assay can be used. For example, a preformed complex of the target gene product and the interactive cell or extracellular binding partner product in which the target gene products or their binding partners are labeled is prepared, but the signal generated by the marker is inactivated due to the complex formation (see, for example, US Patent No. 4,109,496 using this strategy for immunoassays). The addition of a test substance that competes and displaces one of the species of the preformed complex will result in the generation of a signal above the background level. In this way, test substances can be identified that alter the interaction of target gene product-partner of
Union.

In another aspect, 15603 proteins can be used as "bait proteins" in a double hybrid assay or in a triple hybrid assay (see, for example, US Patent No. 5,283,317; Zervos et al . (1993) Cell 72: 223-232; Madura et al . (1993) J. Biol. Chem . 268: 12046-12054. Bartel et al . (1993) Biotechniques 14: 920-924; Iwabuchi et al . (1993) Oncogene 8: 1 693-1696; and Brent document WO 94/10300), to identify other proteins that bind or interact with 15603 ("15603-binding proteins" or "15603-bp") and are involved in the activity of 15603. These 15603 -bp can be signal activators or inhibitors by 15603 proteins or 15603 targets such as, for example, elements downstream of a 15603-mediated signaling path.

The double hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA binding and activation domains. In summary, the assay uses two different DNA constructs. In one construct, the gene that encodes a 15603 protein is fused to a gene that encodes the DNA binding domain of a known transcription factor (eg, GAL-4). In the other construction, a DNA sequence from a library of DNA sequences, which encodes an unidentified protein ("prey" or "sample") is fused to a gene that encodes the activation domain of the known transcription factor. (Alternatively: 15603 protein can be fused to the activator domain.) If the "bait" and "prey" proteins can interact, in vivo, forming a 15603 dependent complex, the DNA binding domains and transcription factor activation domains They get very close. This proximity allows the transcription of an indicator gene (for example, LacZ) that is operably linked to a transcription regulatory site that responds to the transcription factor. Expression of the reporter gene can be detected and colonies of cells containing the functional transcription factor can be isolated and used to obtain the cloned gene encoding the protein that interacts with the 15603 protein.

As an alternative, modulators of the expression of 15603. For example, a cell or a mixture without cells are contacted with a candidate compound and evaluated mRNA or 15603 protein expression with respect to level of mRNA or 15603 protein expression in the absence of the compound candidate. When the expression of mRNA or 15603 protein is higher in the presence of the candidate compound that in his absence, the Candidate compound is identified as a stimulator of the protein or mRNA expression of 15603. Alternatively, when mRNA or 15603 protein expression is lower (less than one statistically significant form) in the presence of the compound candidate that in his absence, the candidate compound is identified as an inhibitor of mRNA or 15603 protein expression. The expression level of mRNA or 15603 protein can be determined by methods described herein to detect mRNA or the 15603 protein.

Also described herein is a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell based assay or a cellless assay, and the ability of the agent to modulate the activity of a 15603 protein can be confirmed in vivo, for example, in an animal such as an animal model for an aberrant or deficient function or expression of an ionic channel.

New agents identified by the screening assays described above are also described herein. An agent identified as described herein (for example, a 15603 modulating agent, an antisense 15603 nucleic acid molecule, a 15603 specific antibody or a 15603 binding partner) can be used in an appropriate animal model for determine the efficacy, toxicity, side effects or mechanisms of action of treatment with said agent. In addition, new agents identified by the screening assays described above may be used for the treatments as described herein.
memory.

The invention is further illustrated by The following example, which should not be considered limiting.

Example Gene Expression Analysis

Total RNA was prepared from various human tissues by a single stage extraction method using  STAT-60 RNA according to the instructions of the manufacturer (TelTest, Inc). Each RNA preparation was treated with DNase I (Ambion) at 37 ° C for 1 hour. It was determined that the DNAse I treatment was completed if the sample required at least 38 cycles of PCR amplification to reach a level fluorescence threshold using β-2 microglobulin as an internal amplicon reference. The integrity of RNA samples after DNase I treatment was confirmed by agarose gel electrophoresis and bromide staining of ethidium After phenol extraction, cDNA was prepared from of the sample using the SUPERSCRIPT ™ Choice System following the manufacturer's instructions (GibcoBRL). For each sample of RNA was simulated transcribed a negative RNA control without reverse transcriptase.

The expression of human 15603 protein is measured by quantitative PCR TaqMan® (Perkin Elmer Applied Biosystems)  in the cDNA prepared from a variety of cell lines and normal and diseased human tissues (for example, cancerous).

Probes were designed by the software PrimerExpress (PE Biosystems) based on the gene sequence of the 15603 human protein. Each 15603 protein gene probe human was labeled using FAM (6-carboxyfluorescein), and the β2-microglobulin reference probe It was labeled with a different fluorescent dye, VIC. This way, the differential marking of the target gene and the reference gene internal allowed measurement in the same well. They were added direct and reverse primers and probes for both the β2-microglobulin as for the target gene at TaqMan® universal PCR master mix (PE Applied Biosystems). Although the final concentration of primer and probe could vary, each one was internally constant within a given experiment. A typical experiment contained a 200 nM concentration of primers direct and inverse plus a 100 nM concentration of probe for β-2 microglobulin and a concentration of 600 nM of direct and inverse primers plus a 200 nM concentration of probe for the target gene. The TaqMan matrix experiments are performed on an ABI PRISM 7700 sequence detection system (PE Applied Biosystems). The conditions of the cycle apparatus The following were thermal: maintenance for 2 min at 50 ° C and 10 min at 95 ° C, followed by a two-stage PCR for 40 cycles 95 ° C for 15 sec followed by 60 ° C for 1 min.

The following method was used to calculate quantitatively the expression of the human 15603 protein gene in the various tissues with respect to the expression of β-2 microglobulin in the same tissue. He threshold cycle value (Ct) is defined as the cycle to which detects a statistically significant increase in fluorescence. A lower value of Ct is indicative of a higher concentration of MRNA The Ct value of the human 15603 protein gene is normalizes by subtracting the Ct value of the gene from the β-2 microglobulin to obtain a value of ΔCt using the following formula: ΔCt = human Ct_ { 59914 and 59921 - Ct? -2 microglobulin. The expression It is then calibrated against a cDNA sample showing a level comparatively low protein gene expression 15603 human The value of ΔCt for the calibrator sample then subtract from? Ct for each tissue sample of according to the following formula: ΔCt = ΔCt -_ {sample} - Ct -_ {calibrator} \ cdot Then calculate the relative expression using the arithmetic formula given by 2 ^ -? Delta Ct. Subsequently it is represented graphically expression of the target human 15603 protein gene in each of the tissues tested as described in more detail more ahead.

The results indicate an expression significant of 15603 in hemangioma, vascular smooth muscle, megakaryocytes, Wilm tumor; at medium levels in cerebral cortex normal, proliferative endothelial uterus and cell; and at levels low in adipose tissue, normal human internal mammary artery, diseased human aorta, diseased human artery, diseased human vein and normal human vein, normal heart, CHF heart, normal kidney, skeletal muscle, pancreas, primary osteoblasts, hypothalamus brain, normal breast, normal ovary, normal prostate, tumor of prostate, salivary glands, normal colon, colon tumor, lung normal, lung tumor, pulmonary COPD, colon IBD, normal spleen, normal tonsil, normal small intestine, pressure ulcer in the skin, synovium, glioblastomas, fetal adrenal gland, liver Fetal and fetal heart.

       \ vskip1.000000 \ baselineskip
    

       \ vskip1.000000 \ baselineskip
    

TABLE 1 Phase 1.5.1 Expression of 15603 with beta 2

one

2

3

       \ vskip1.000000 \ baselineskip
    

TABLE 2 15603 Expression in Glasses Panel 2.1

4

5

6

       \ vskip1.000000 \ baselineskip
    

       \ vskip1.000000 \ baselineskip
    

TABLE 3 1506 expression

7

8

9

       \ vskip1.000000 \ baselineskip
    

       \ vskip1.000000 \ baselineskip
    

TABLE 4 Expression of 15603 with β2 in the Panel Angiogenesis

10

Claims (8)

1. A method to identify a capable compound of treating an angiogenic disorder, which includes: i) contacting a candidate compound with the nucleic acid molecule of SEQ ID NO: 3 as shown in figure 1; Y ii) test the ability of the compound to inhibit the expression of the nucleic acid molecule, identifying in this way a compound capable of treating an angiogenic disorder. 2. A method to identify a capable compound of treating an angiogenic disorder, which includes: i) contacting a candidate compound with a polypeptide encoded by the nucleic acid molecule of the SEQ ID NO: 3 as shown in Figure 1 or a cell expressing the polypeptide encoded by the nucleic acid molecule of the SEQ ID NO: 3 as shown in figure 1; Y ii) test the ability of the compound to inhibit the ion channel activity of the polypeptide, identifying in this way a compound capable of treating an angiogenic disorder. 3. A method according to claim 2, wherein said assay is a cell based assay and said Candidate compound contacts a cell that expresses said polypeptide. 4. A method according to claim 2, wherein said assay is a test without cells. 5. A method according to claim 4, wherein said polypeptide is provided as an ionic channel in a plasma membrane 6. A method according to claim 5, wherein said test of the ability of the compound to inhibit the ionic channel activity of the polypeptide comprises measuring the ion flow through said ion channel. 7. A method according to claim 6, wherein the ion flow through said ion channel is measured by zonal grip. 8. A method according to any one of claims 1 to 7, further comprising confirming the ability of a test compound to inhibit the expression of the nucleic acid molecule of SEQ ID NO: 3 as shown in the Figure 1 or inhibit the polypeptide encoded by the molecule nucleic acid of SEQ ID NO: 3 as shown in Figure 1 in An animal model.